Mediators of signal transduction

ABSTRACT

Polypeptides capable of regulating signal transduction, which preferably exhibit kinase activity, or antibodies against such polypeptides that inhibit the interaction of these polypeptides with other mediators of signal transduction, may be used in the identification, prevention or treatment of disease, preferably cardiac disease, in mammalian hosts. In addition, these polypeptides can facilitate the identification or isolation of additional mediators of signal transduction associated with disease, preferably cardiac disease, which in turn may also be used in the identification, prevention or treatment of disease, preferably cardiac disease, in mammals.

RELATED APPLICATIONS

[0001] This application is a divisional application of U.S. patent application Ser. No. 9/548,473, filed Apr. 13, 2000, now U.S. Pat. No. ______, and claims the benefit of priority from U.S. Provisonal Patent Application No. 60/129,552, filed Apr. 16, 1999, both of which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to compounds and methods for the identification, prevention or treatment of disease, preferably cardiac disease, in a mammal through the administration of polypeptides capable of regulating signal transduction, which preferably exhibit kinase activity, or antibodies against such polypeptides that inhibit the interaction of these polypeptides with other mediators of signal transduction. In addition, the compounds and methods of the present invention can facilitate the identification or isolation of additional mediators of signal transduction associated with disease, preferably cardiac disease, which in turn may also be used in the identification, prevention or treatment of disease, preferably cardiac disease, in mammals.

BACKGROUND OF THE INVENTION

[0003] Certain biological functions, such as growth and differentiation, are tightly regulated by signal transduction pathways within cells. Signal transduction pathways maintain the balanced steady state functioning of a cell. Disease states can arise when signal transduction in a cell breaks down, thereby removing the control that typically exists over cellular functions. Because signal transduction networks regulate a multitude of cellular functions depending upon the cell type, a wide variety of diseases can result from abnormalities in such networks. Devastating diseases such as cancer, autoimmune diseases, allergic reactions, inflammation, neurological disorders and hormone-related diseases can result from abnormal signal transduction. For example, tumors may develop when regulation of cell growth is disrupted.

[0004] Despite a long-felt need to understand and discover methods for regulating cells involved in various disease states, the complexity of signal transduction pathways has precluded the development of products and processes for regulating cellular function by manipulating signal transduction pathways in a cell. As such, there remains a need for products and processes that permit the implementation of predictable controls of signal transduction in cells, thus enabling the treatment of various diseases that are caused by abnormal cellular function.

[0005] Such diseases may include cardiac diseases, which may include congestive heart failure (CHF), dilated congestive cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, mitral valve disease, aortic valve disease, tricuspid valve disease, angina pectoris, myocardial infarction, cardiac arrhythmia, pulmonary hypertension, arterial hypertension, renovascular hypertension, arteriosclerosis, atherosclerosis, and cardiac tumors. By way of example, CHF is a major cardiac disease associated with extensive morbidity and mortality. Approximately five million individuals in the United States suffer from some form of CHF. Traditionally, treatment of CHF occurs by a series of agents including diuretics, vasodilators, angiotensin converting enzyme inhibitors, B-adrenergic antagonists, and positive inotropes like digoxin. These drugs, however, principally provide symptomatic relief and typically only extend the life of one suffering from the disease for periods ranging from 6-12 months.

[0006] The pathophysiology of CHF is rather complex. Generally, the central hallmark of the disease is the inability of the heart to pump sufficient oxygenated blood to meet the demands of peripheral tissues. Numerous etiologies contribute to the development of CHF, including primary diseases of, or insults to, the myocardium itself, cardiac defects, hypertension, inflammation, kidney disease and vascular disease. These conditions lead to the hypertrophy and remodeling of the cardiac ventricles which, if unchecked, ultimately reduce the mechanical performance of the heart. Forces associated with the inability of the heart to pump blood ultimately lead to the release of neurohormones like catecholamines, renin-angiotensin, aldosterone, endothelin and related factors into the circulation. Elevations in plasma levels of many of these circulating neurohormones have a deleterious impact on the outcome of patients with CHF. Local production of these neurohormonal factors in the heart is believed to contribute centrally to the disease. Thus, an important therapeutic strategy has been to block this neurohormonal axis contributing to the pathogenesis of this disease.

[0007] Factors known to contribute centrally to the pathophysiology of heart disease are biosynthesized in the heart itself. These factors are produced in cardiac myocytes, fibroblasts, smooth muscle and endothelial cells, and inflammatory cells associated with the myocardium. For example, the heart contains its own renin-angiotensin system. Blockade of the cardiac renin-angiotensin system may contribute significantly to the therapeutic efficacy of the therapeutic class of agents known as angiotensin converting enzyme (ACE) inhibitors.

[0008] The heart also produces other factors including endothelins, bradykinin, adrenomedullin, tumor necrosis factor, transforming growth factors, and natriuretic peptides. Unfortunately, therapeutic strategies are limited to the modulation of such substances, which are already known to contribute to the disease. Indeed, the functional contributions of only a minor fraction of all known secreted factors encoded by the human genome have apparently been defined.

[0009] The foregoing shows a need for methods and products involving the prevention or treatment of disease in mammals involving the mediation of signal transduction. The administration of polypeptides capable of regulating signal transduction, which preferably exhibit kinase activity, or antibodies against such polypeptides that inhibit the interaction of these polypeptides with other mediators of signal transduction, in addition to the identification or isolation of additional mediators of signal transduction associated with disease, preferably cardiac disease, which in turn may also be used in the identification, prevention or treatment of disease, preferably cardiac disease, in mammals, can facilitate such prevention or treatment.

SUMMARY OF THE INVENTION

[0010] An objective of the present invention is therefore the prevention or treatment of disease, preferably cardiac disease, in mammals through the administration of polypeptides capable of regulating signal transduction, which preferably exhibit kinase activity, or antibodies against such polypeptides that inhibit the interaction of these polypeptides with other mediators of signal transduction, in addition to the identification or isolation of additional mediators of signal transduction associated with disease, preferably cardiac disease, which in turn may also be used in the identification, prevention or treatment of disease, preferably cardiac disease, in mammals.

[0011] In accomplishing these and other objectives, the present invention preferably provides a purified polypeptide comprising the amino acid sequence of SEQ ID NOS: 1, 4, 6, 7, 8 or 9 wherein the polypeptide is capable of regulating signal transduction. In a preferred embodiment, the polypeptide is capable of catalyzing the transfer of a phosphate group from a donor molecule to an acceptor molecule.

[0012] In another embodiment, the present invention preferably provides an isolated DNA molecule encoding a purified polypeptide comprising the amino acid sequence of SEQ ID NOS: 1, 4, 6, 7, 8 or 9 wherein the polypeptide is capable of regulating signal transduction. The present invention may also preferably be an isolated DNA molecule comprising the nucleotide sequence of SEQ ID NOS: 2, 3 or 5.

[0013] In yet another embodiment, the present invention preferably provides a vector comprising a DNA molecule encoding a purified polypeptide comprising the amino acid sequence of SEQ ID NOS: 1, 4, 6, 7, 8 or 9 wherein the polypeptide is capable of regulating signal transduction. In another aspect, the present invention provides a host cell transformed with such a vector. In one other embodiment, the present invention may preferably provide the above-described transformed host cell, where the host cell produces a polypeptide capable of regulating signal transduction. In a preferred embodiment, the above-described transformed host cell produces a polypeptide capable of catalyzing the transfer of a phosphate group from a donor molecule to an acceptor molecule.

[0014] In another preferred embodiment, the present invention provides an isolated antibody against a polypeptide comprising the amino acid sequence of SEQ ID NOS: 1, 4, 6, 7, 8 or 9 wherein the polypeptide is capable of regulating signal transduction. In a preferred embodiment, the antibody is a monoclonal antibody. In another preferred embodiment, the antibody is capable of inhibiting the regulation of signal transduction. In yet another preferred embodiment, the antibody is capable of inhibiting the transfer of a phosphate group from a donor molecule to an acceptor molecule.

[0015] The present invention may also preferably provide an isolated nucleic acid capable of hybridizing under high stringency conditions to a DNA molecule comprising the nucleotide sequence of SEQ ID NOS: 2, 3 or 5. In a preferred embodiment, this isolated nucleic acid is capable of inhibiting the regulation of signal transduction. In yet another preferred embodiment, this isolated nucleic acid is capable of inhibiting said transfer of said phosphate group from said donor molecule to said acceptor molecule.

[0016] In a preferred embodiment, the present invention provides a method of preventing or treating disease in a mammal comprising administering to said mammal an effective amount of material, selected from the group consisting of the polypeptide comprising the amino acid sequence of SEQ ID NOS: 1, 4, 6, 7, 8 or 9 wherein the polypeptide is capable of regulating signal transduction, and the antibody against this polypeptide, in a pharmaceutically acceptable sterile vehicle. In a preferred embodiment, the mammal may be a human. In another, the disease may be cardiac disease.

[0017] The present invention may also preferably provide a vaccine for preventing disease in a mammal comprising administering to said mammal an effective amount of material, selected from the group consisting of the polypeptide comprising the amino acid sequence of SEQ ID NOS: 1, 4, 6, 7, 8 or 9 wherein the polypeptide is capable of regulating signal transduction, and the antibody against this polypeptide, in a pharmaceutically acceptable sterile vehicle. In a preferred embodiment, the mammal may be a human. In another, the disease may be cardiac disease.

[0018] In a preferred embodiment, the present invention provides a method of preventing or treating disease in a mammal comprising administering to said mammal syngeneic cells transformed with a vector comprising a DNA molecule encoding a purified polypeptide comprising the amino acid sequence of SEQ ID NOS: 1, 4, 6, 7, 8 or 9 wherein the polypeptide is capable of regulating signal transduction, and wherein the transformed syngeneic cells produce a polypeptide capable of regulating signal transduction. In a preferred embodiment, the mammal may be a human. In another, the disease may be cardiac disease.

[0019] The present invention may also preferably provide a method of preventing or treating disease in a mammal comprising administering to said mammal syngeneic cells transformed with a vector comprising a DNA molecule encoding a purified polypeptide comprising the amino acid sequence of SEQ ID NOS: 1, 4, 6, 7, 8 or 9 wherein the polypeptide is capable of regulating signal transduction, and wherein the transformed syngeneic cells produce a polypeptide capable of catalyzing the transfer of a phosphate group from a donor molecule to an acceptor molecule. In a preferred embodiment, the mammal may be a human. In another, the disease may be cardiac disease.

[0020] In a preferred embodiment, the present invention may provide a kit for detecting the expression of a protein capable of regulating signal transduction, comprising a polypeptide, which comprises the amino acid sequence of SEQ ID NOS: 1, 4, 6, 7, 8 or 9 wherein the polypeptide is capable of regulating signal transduction. In a preferred embodiment, this kit further comprises a detectable label selected from the group consisting of calorimetric, enzymatic, chemiluminescent, fluorescent and radioactive labels.

[0021] In another preferred embodiment, the present invention may provide a kit for detecting the expression of a protein capable of acting as a donor molecule or an acceptor molecule of a phosphate group comprising a polypeptide, which comprises the amino acid sequence of SEQ ID NOS: 1, 4, 6, 7, 8 or 9 wherein the polypeptide is capable of regulating signal transduction. In a preferred embodiment, this kit further comprises a detectable label selected from the group consisting of calorimetric, enzymatic, chemiluminescent, fluorescent and radioactive labels.

[0022] The present invention may also preferably provide a method for detecting the expression of a protein capable of regulating signal transduction, comprising contacting a sample with a polypeptide, which comprises the amino acid sequence of SEQ ID NOS: 1, 4, 6, 7, 8 or 9 wherein the polypeptide is capable of regulating signal transduction, and detecting any effect of the sample on an indicator of signal transduction. In a preferred embodiment, the polypeptide is immobilized to a solid support. In another preferred embodiment, the phosphate group is detectably labeled.

[0023] In another preferred embodiment, the present invention may provide a method for detecting the expression of a protein capable of acting as a donor molecule or an acceptor molecule of a phosphate group, comprising contacting a sample with a polypeptide, which comprises the amino acid sequence of SEQ ID NOS: 1, 4, 6, 7, 8 or 9 wherein the polypeptide is capable of regulating signal transduction, and detecting any effect of the sample on an indicator of signal transduction, and detecting any transfer of the phosphate group. In a preferred embodiment, the polypeptide is immobilized to a solid support. In another preferred embodiment, the phosphate group is detectably labeled.

[0024] Other objectives, features, and advantages of the present invention will become apparent from the following detailed description. The detailed description and the specific examples, while indicating preferred embodiments of the invention, are provided by way of illustration only. Accordingly, the present invention also includes those various changes and modifications within the spirit and scope of the invention that may become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

[0025]FIG. 1 illustrates eight overlapping human cDNA clones of 19G5.

[0026]FIG. 2 depicts homology alignment of amino acid sequences of human, rat and mouse clones corresponding to 19G5.

[0027]FIG. 3 depicts the likely gene structure of human 19G5 (H19G5), which reveals at least 10 exons and 9 introns.

[0028]FIG. 4 is a schematic diagram of four cDNA clones corresponding to splicing variants of H19G5. The longest clone (C11) contains two kinase domains, the N-terminal and the C-terminal kinase domains (N-KD and C-KD).

[0029]FIG. 5 is a schematic diagram comparing the domain structure of 19G5 and Trio proteins.

[0030]FIG. 6 is the sequence comparison of the kinase domains of H19G5 (N-terminal and C-terminal kinase domains), Trio, and smooth muscle myosin light chain (SM MLCK).

[0031]FIG. 7 is the sequence comparison of the guanine nucleotide exchange factor (GEF) domains of H19G5 and Trio.

[0032]FIG. 8 shows the subcellular localization of 19G5-GFP fusion proteins in mouse myoblast cell line C2C12. Three 19G5-GFP fusion protein expression constructs were made using three different 19G5 cDNA clones, the longest human 19G5 clone C11 [h19G5(C11)-GFP], a 2.7 kb clone of human 19G5 containing the C-terminal kinase domain [h19G59F1)-GFP], and the rat 19G5 small transcript [r19G5(S)-GFP]. The control GFP vector and the 19G5-GFP fusion expression constructs were transfected into C2C12 cells. The 19G5-GFP fusion proteins' localization was detected using confocal microscopy.

[0033]FIG. 9 shows that H19G5 protein expressed by the clone C11 binds to the small G protein Cdc42. The lysate of 293 EBNA cells transfected with H19G5-C11 expression construct was incubated with GST-Cdc42 immobilized on glutathione-agarose. After washing, the complex was resuspended in SDS sample buffer, boiled and run on a SDS-PAGE, and Western blotted with an anti-H19G5 monoclonal antibody.

[0034]FIG. 10 shows phase contrast micrographs of C2C12 myoblasts undergoing differentiation into myotubes. Undifferentiated C2C12 cells are shown in Day 0. Differentiation of C2C12, induced by placing in 2% horse serum, is apparent on Day 3 and Day 5 as shown.

[0035]FIG. 11 shows induction of myogenin protein expression when C2C12 myoblast cells are induced to differentiate into myotubes. Myogenin is a marker of myotubes.

[0036]FIG. 12 shows induction of 19G5 RNA expression during the differentiation of myoblasts into myotubes.

[0037]FIG. 13 shows inhibition of the induction of 19G5 expression by TFG-β during C2C12 differentiation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Those skilled in the art will recognize that the products and methods embodied in the present invention may be applied to a variety of systems, constructed with various materials using various methods. Accordingly, the present invention is not limited to any particular environment, and the following description of specific embodiments of the present invention are for illustrative purposes only.

[0039] The present invention preferably provides methods for the prevention or treatment of disease, preferably cardiac disease, in mammals through the administration of polypeptides capable of regulating signal transduction, which preferably exhibit kinase activity, or antibodies against such polypeptides that inhibit the interaction of these polypeptides with other mediators of signal transduction, in addition to the identification or isolation of additional mediators of signal transduction associated with disease, preferably cardiac disease, which in turn may also be used in the identification, prevention or treatment of disease, preferably cardiac disease, in mammals. The cardiac diseases according to the present invention may include congestive heart failure (CHF), dilated congestive cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, mitral valve disease, aortic valve disease, tricuspid valve disease, angina pectoris, myocardial infarction, cardiac arrhythmia, pulmonary hypertension, arterial hypertension, renovascular hypertension, arteriosclerosis, atherosclerosis, and cardiac tumors.

[0040] An embodiment of the invention is a purified polypeptide comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NOS: 1, 4, 6, 7, 8 or 9. As used herein, polypeptide refers to a linear series of amino acid residues connected to one another by peptide bonds between the alpha-amino groups and carboxy groups of adjacent amino acid residues. Additional covalent bonds between portions of the peptide may also be present to restrain the conformation of the molecule, such as amide and disulfide bonds. When used herein, polypeptide also refers to a linear series of amino acid residues connected one to the other as in a peptide. The term synthetic peptide means a chemically derived chain of amino acid residues linked together by peptide bonds that is free of naturally occurring proteins and fragments thereof.

[0041] The one and three-letter symbols used to represent the amino acid residues in the polypeptides of the present invention are those symbols commonly used in the art. The amino acid residues are preferred to be in the L isomeric form. However, residues in the D isomeric form may be substituted for any L-amino acid, as long as the desired functional property of signal transduction mediation is retained by the peptide. The one and three-letter symbols used herein refer to the following amino acids: Ser (S) is serine; Ile (I) is isoleucine; Gln (Q) is glutamine; Phe (F) is phenylalanine; His (H) is histidine; Trp (W) is tryptophan; Lys (K) is lysine; Asn (N) is asparagine; Leu (L) is leucine; Gly (G) is glycine; Thr (T) is threonine; Asp (D) is aspartic acid; Arg (R)is arginine; and Ala (A) is alanine.

[0042] Polypeptides of the present invention include variants, fragments and chemical derivatives of the polypeptides comprising the amino acid sequence of SEQ ID NOS: 1, 4, 6, 7, 8 or 9 as long as they are capable of mediating signal transduction. Polypeptides thus may include soluble peptides, Ig-tailed fusion peptides (including immunoadhesions), members of random peptide libraries (see, e.g., Lam, K. S. et al., Nature 354:82-84 (1991); Houghten, R. et al., Nature 354:84-86 (1991)), combinatorial chemistry-derived molecular libraries made of D-and/or L-configuration amino acids, and phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang, Z. et al., Cell 72:767-778(1993)).

[0043] Polypeptides of the present invention may also include polypeptides that can be isolated from nature or can be produced by recombinant and/or synthetic means. Such native sequence polypeptides specifically refers to naturally-occurring truncated or secreted forms (e.g., an extracellular domain sequence), as well as naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants of the named polypeptides.

[0044] The term variant refers to any polypeptide having an amino acid sequence, in comparison to the amino acid sequences of the polypeptides of the present invention, in which one or more amino acids have been substituted with other amino acids; where the substituted amino acids allow or require the polypeptide to assume the equilibrium conformation of the domain of the parent protein. Often, cysteine, lysine and glutamic acid will be used for their side chains which can form covalent linkages to restrict the conformation of a peptide. The term variant refers to any polypeptide in which one or more amino acids are added and/or substituted and/or deleted and/or inserted at the N- or C-terminus or anywhere within the corresponding native sequence, and which retains signal transduction mediation activity of the corresponding native polypeptide. The variants herein preferably comprise a sequence that has at least about 80% sequence identity, more preferably at least about 85% sequence identity, even more preferably at least about 90% sequence identity, most preferably at least about 95% sequence identity, with the amino acid sequence of SEQ ID NOS: 1, 4, 6, 7, 8 or 9.

[0045] In such amino acid sequences, one or more amino acids in the fundamental sequence may preferably be substituted with another amino acid(s), the charge and polarity of which is similar to that of the native amino acid, i.e., a conservative amino acid substitution, resulting in a silent change. Substitutes for an amino acid within the fundamental polypeptide sequence can be selected from other members of the class to which the naturally occurring amino acid belongs. Amino acids can be divided into the following four groups: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids; and (4) neutral non-polar amino acids. Representative amino acids within these various groups include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cyteine, cystine, tyrosine, asparagine, and glutamine; (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.

[0046] The term variant shall also include any polypeptide having one or more amino acids deleted from or added to an amino acid sequence of a mediator of signal transduction, but which still retains signal transduction mediation activity. The term fragment shall refer to any shorter version of the polypeptides herein, wherein the fragment is capable of mediating signal transduction.

[0047] Sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a native polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. The % sequence identity values are preferably generated by the NCBI BLAST2.0 software as defined by Altschul et al., (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res., 25:3389-3402. The parameters are set to default values, with the exception of the Penalty for mismatch, which is set to −1. Other algorithms, such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Groups, 575 Science Dr., Madison, Wis.), are also suitable. The selection of the non-default parameters to achieve maximum sequence identity is well within the skill of a person skilled in the art.

[0048] Antibodies of the present invention may include any polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab′)₂ and FAb fragments, and epitope-binding fragments thereof.

[0049] Without further elaboration, one skilled in the art with the preceding description can utilize the present invention to its fullest extent. The following examples are illustrative only, and not intended to limit the remainder of the disclosure in any way.

EXAMPLE 1 Isolation and Characterization of 19G5 Clones from Rat, Mouse and Human

[0050] Isolation of Partial and Full-Length 19G5 Clones from Rat, Mouse and Human

[0051] Microarray technology was used to identify genes that are differentially expressed in normal and diseased rat heart. A sequence designated as 19G5 was down regulated in 12-week myocardial infarct (MI) rat hearts. A rat cDNA clone corresponding to 19G5 (R19G5) was isolated and nucleotide sequence determined. The deduced amino acid sequence of the clone revealed homology to the catalytic domain of kinases, thus suggesting that the protein product of R19G5 might be involved in signal transduction. A hybridization of multiple tissue Northern blot with the R19G5 probe showed that the gene is highly expressed in heart as a 2 kb and 4.4 kb transcripts (FIG. 8).

[0052] A full length cDNA for the 2 kb R19G5 transcript was cloned using 5′ RACE technique. The R19G5 has an open reading frame of 1644 base pairs which encodes a protein of 548 amino acids (SEQ ID NO: 8)

[0053] A mouse 19G5 (M19G5) EST clone was identified by searching the EST database. Northern blot hybridization using the M19G5 EST clone as probe detected a major transcript of 2 kb in heart. There is also a low level of expression in lung. The smeary hybridization was also detected as in the rat and human but was less prominent. M19G5 was also expressed in 17-day old mouse embryos suggesting that it may play a role in embryo development. Sequence analysis of the M19G5 clone showed that it is 1900 base pairs long and has an open reading frame of 1644 base pairs coding for a polypeptide of 548 amino acids (SEQ ID NO: 9), suggesting that it likely represents full length 2 kb transcript.

[0054] A number of human cDNA clones corresponding to 19G5 (H19G5) were isolated from human CDNA library using R19G5 as a probe. FIG. 1 shows eight overlapping cDNA clones of H19G5. Extensive overlap among these clones helped build a consensus nucleotide sequence (SEQ ID NO: 2) designated as H19G5 contig. The sequence corresponds to a major transcript (˜5 kb) expressed in human heart. The sequence of the contig revealed that it is complete at the 3′-end since it contains a polyadenylation signal (AATAAA) as well as polyA residues at the 3′-end. It has a potential open reading frame, coding for 1351 amino acid residues, that extends beyond the 5′-end of the contig indicating that it is incomplete at the 5′-end. The deduced amino acid sequence (SEQ ID NO: 1) revealed a protein kinase domain at the C-terminal end (amino acid residues 1056 to 1309) and also a partial protein kinase domain towards the N-terminal end (amino acid residues 1 to 105) of this truncated clone. The sequence information of this contig (SEQ ID NO: 2) was used to devise antisense primers corresponding to the 5′-end, which were used in 5′ RACE (rapid amplification of cDNA ends) to isolate cDNA clones with longer inserts. A cDNA clone containing full-length coding sequence was isolated. The nucleotide sequence of the clone (SEQ ID NO: 3) revealed an open reading frame that could potentially code for 1667 amino acids long full-length polypeptide (SEQ ID NO: 4).

[0055] Using the 5′ RACE technique, the full coding region for one of the larger H19G5 (3 kb or 5 kb) transcripts was cloned. It has an open reading frame of 2613 base pairs encoding a protein of 811 amino acids.

[0056]FIG. 2 shows the amino acid sequence alignment of the three full-length 19G5 proteins. Both the R19G5 (SEQ ID NO: 8) and M19G5 (SEQ ID NO: 9) proteins overlap with the C-terminal two-third of a splicing variant of the H19G5 protein (SEQ ID NO: 7). The identity between the R19G5 and M19G5 proteins is 97%. The three proteins are 85% identical in their sequences.

[0057] In order to understand the genomic organization of H19G5 gene, phage genomic library was screened and eleven H19G5 genomic clones were isolated. Restriction enzyme mapping of these clones detected no polymorphism, suggesting that H19G5 may be a single copy gene. The entire genomic DNA region encompassing H19G5 was sequenced and found to contain at least 10 exons and 9 introns as shown in FIG. 3.

[0058] One of the H19G5 cDNA clones contained deletion of two amino acid residues (alanine and proline) in the kinase domain. Sequence analysis of the genomic H19G5 DNA revealed an intron located immediately upstream of the alanine residue. There are two adjacent splicing acceptor sites that are four nucleotides apart at the 3′ end of the intron. A splicing event using the first acceptor site generates a protein that includes the two residues, alanine and proline. Utilization of the second splicing acceptor site creates a H19G5 protein with the two amino acids deleted in its kinase domain. These two residues are highly conserved among all kinases. This deletion form of H19G5 protein may thus exhibit reduced or no kinase activity at all, and may serve as a dominant negative inhibitor of the kinase activity of H19G5. This provides a possible mechanism to regulate the activity of H19G5 in vivo.

[0059] Isolation of cDNA Clones of Splicing Variants of H19G5

[0060] Multiple transcripts were detected in Northern analysis of human and rat cells, particularly in heart tissues (As shown in FIGS. 8 and 9). However, the restriction analysis of the genomic clones and Southern analysis of genomic DNA suggested single-copy nature of H19G5 gene. These observations indicated the possibility of alternative splicing as a source of multiple transcripts. Four cDNA clones representing various splicing variants were isolated and are schematically shown in FIG. 4. Complete nucleotide sequence (SEQ ID NO: 5) of the longest clone C11 was determined and the amino acid sequence of a large open reading frame contained therein was deduced (SEQ ID NO: 6). The large ORF has a potential to encode a protein of 2596 amino acid residues. The sequence analysis revealed the presence of a number of structurally and functionally important domains in H19G5. The presence of some of the domains strengthens the possibility of the involvement of H19G5 in signal transduction. For example, H19G5 has two kinase domains, one at the N-terminal (1094 to 1351 amino acid residues, N-KD) and the other at the C-terminal (2301 to 2553 amino acid residues, C-KD) end. H19G5 also has a Guanine nucleotide Exchange Factor (GEF) domain (325 to 504 amino acid residues), and a Pleckstrin Homology (PH) domain (532 to 634 amino acid residues). Additionally, H19G5 contains five immunoglobulin (Ig) like domains distributed throughout the sequence.

[0061] Structural and Functional Features of Full-Length H19G5 Protein Sequence

[0062] The full-length H19G5 amino acid sequence (SEQ ID NO: 6) shows sequence similarity to some functionally important domains of a protein called Trio. The comparison of the domain structure of H19G5 and Trio is schematically depicted in FIG. 5. Both kinase domains of H19G5 (N-KD and C-KD) are homologous to a single kinase domain of Trio as well as to a kinase domain of smooth muscle myosin light chain kinase (SM MLCK) as shown in FIG. 6. The identity between H19G5 N-KD and Trio KD is about 40% and between H19G5 N-KD and SM MLCK is about 38%. The sequence identity between the two kinase domains of H19G5 is about 30%. A single GEF domain of H19G5 is homologous to both GEF domains (GEF-D 1 and GEF-D2) of Trio as shown in FIG. 7.

[0063] Proteins containing GEF domains are involved in signal transduction (for a review, see Cherfils and Chardin, Trends Biochem. Sci. 24: 306-311[1999]). GEF domains promote exchange of GTP for GDP on GTP-binding proteins (G proteins) and thereby positively regulate their activities. As these proteins harbor intrinsic GTPase activity, they are also referred to as GTPases. These small G proteins, as opposed to trimeric G proteins, belong to a superfamily of Ras-like proteins. These proteins are bound to the inner face of plasma membrane, and usually exist in GDP-bound “inactive” state. When a ligand interacts with a membrane bound receptor, alteration of conformation allows the receptor to interact with a G protein. This interaction results in conformational change in the G protein that weakens the affinity for GDP and leads to replacement of GDP with GTP. This nucleotide exchange is greatly accelerated or promoted by proteins containing GEF domains. Once bound to GTP, the G proteins assume an “active” state in which they interact with the downstream effectors and facilitate transduction of signal from membrane to the nucleus. However, the activity of the G proteins is tightly controlled as their intrinsic GTPase activity rapidly hydrolyzes bound GTP into GDP and restores “inactive” status. Thus, G proteins function as molecular switches in signal transduction. A number of membrane receptors operate through G proteins. The downstream effectors of the activated G proteins include various protein kinases constituting a cascade of protein phosphorylation that brings about a desired change in gene expression.

[0064] As discussed in the preceding section, GEF domains play a critical role in signal transduction by controlling the activation of G proteins. Trio is a complex protein possessing two GEF domains, each with adjacent pleckstrin homology (PH) domains and Src Homology-3 (SH3) domains, a protein serine/threonine kinase domain with an adjacent immunoglobulin-like domain and multiple spectrin-like domains (Medley et al., J. Cell Sci. 112: 1825-1834[1999]). Trio CDNA clone was isolated by virtue of its ability to interact with protein tyrosine phosphatase (PTP) domain of a protein called LAR (Debant et al., Proc. Natl. Acad. Sci. USA 93: 5466-5471[1996]). LAR is a broadly expressed transmembrane protein tyrosine phosphatase comprised of a cell adhesion-like extracellular region and two intracellular PTPase domains, and is proposed to regulate cell-matrix interactions (Mourey and Dixon, Curr. Opin. Gen. Dev. 4: 31-39[1994]). Trio represents a unique member of the Rho-GEFs family possessing two functional GEF domains of distinct specificities. For example, GEF1 is specifically active on Rac1 GTPase, while GEF2 targets RhoA GTPase (Debant et al, supra). This unique feature allows Trio to link Rho and Rac specific signalling pathways in vivo.

[0065] The Rho family of Ras-like GTPases includes Rac (1, 2 and 3), RhoG, Cdc42Hs, TC10, TTF/RhoH, Rho (A, B and C), RhoD, RhoE, and RhoL. These proteins and other Ras-like proteins constitute Ras superfamily of structurally and functionally related GTPase proteins. These proteins are involved in diverse physiological functions such as control of cell shape (reviewed in Tapon and Hall, Curr. Opin. Cell Biol. 9: 86-92[1997]), cell motility (Aepfelbacher et al., Proc. Natl. Acad. Sci. USA 91: 4263-4267[1994]; and Curr. Biol. 6: 70-75[1996]), cell polarity (Adams et al, J. Cell Biol. 111: 131-142[1990]), smooth muscle contraction (Hirata et al., J. Biol. Chem. 267: 8719-8722[1992]), cell adhesion (Nobes and Hall, Cell 81: 53-62[1995]; Braga et al., J. Cell Biol. 137: 1421-1431 [1997]), cell division (Dutartre et al., J. Cell Sci. 109:367-377[1996]), vesicular transport between organelles such as receptor-mediated endocytosis (Lamaze et al., Nature 382: 177-179[1996]), apoptosis (Esteve et al., Oncogene 11: 2657-2665[1995]; Jimenez et al., Oncogene 10: 811-816[1995]; Gulbins et al., J. Biol Chem. 271: 26389-26394[1996]; Moorman et al., J. Immunol. 156: 4146-4153[1996]; Brenner et al., J. Biol. Chem. 272: 22173-22181[1997]) and normal and pathological cell proliferation (Olson et al., Science 269: 1270-1272[1995]; Hirai et al., J. Biol. Chem. 272: 13-16[1997]; Khosravi-Far et al., Mol. Cell. Biol. 16: 3923-3933[1996]; Qiu et al., Mol. Cell. Biol. 17: 3449-3458[1997]; Roux et al., Curr. Biol. 7: 629-637[1997]).

[0066] The presence of a GEF domain and protein kinase domains along with its homology to Trio suggests that H19G5 may possess guanine nucleotide exchange factor activity and protein kinase activity, both of which are shared by a number of proteins involved in signal transduction.

[0067] Expression Pattern of 19G5 in Rat and Human Tissues

[0068] Northern blot analysis revealed that R19G5 gene is highly expressed in heart as a 2 kb and a 4.4 kb transcripts (FIG. 8). Hybridization was performed using multiple tissue Northern blot (Clontech, Palo Alto, Calif.) and ExpressHyb solution following the manufacturer's protocol. The R19G5 probe also hybridized to mRNAs from skeletal muscle and detected multiple weak bands. High background observed in a lane corresponding to skeletal muscles is not due to RNA degradation as probing of the same blot with β-actin probe detected the right sized transcripts with a clean background (FIG. 8). The significance of the high background in skeletal muscle is not clear. Expression of R19G5 was not detected in brain, kidney, spleen, lung, liver, and testis.

[0069] Hybridization of human multiple tissue Northern blot (Clontech, Palo Alto, Calif.) with H19G5 probe detected a strong transcript of about 5 kb and three minor transcripts of about 3 kb, 2.4 kb, and 1.8 kb in heart tissue (FIG. 9). There were multiple transcripts and high background in skeletal muscle as seen with rat tissue. Only the 3 kb transcript was detected in human fetal heart. There is a low level expression of the 2.4 kb transcript in brain. H19G5 expression was not detected in spleen, lung, liver, kidney, pancreas, thymus, prostates, testis, ovary, small intestine, colon, peripheral blood leukocyte, stomach, thyroid, spinal cord, lymph node, trachea, adrenal gland, bone marrow, uterus muscle, or bladder muscle by Northern blot.

[0070] In order to determine whether the multiple transcripts of 19G5 in human heart are derived from alternative splicing or multiple gene copies, Southern blot analysis of genomic DNA was performed with H19G5 probe (FIG. 10). The results suggest that H19G5 is a single copy gene. This is consistent with the lack of detection of polymorphism in the restriction analysis of the genomic clones of H19G5.

[0071] Functional Characterization of H19G5 Protein

[0072] A 20 amino acid peptide from the C-terminus of H19G5 protein (SEQ ID NO: 1) was used to raise polyclonal antibodies in rabbits. Affinity purified rabbit anti-H19G5 antiserum has been obtained. Two different GST-H19G5 fusion proteins were used as antigens to generate mouse monoclonal anti-H19G5 antibodies. One of the antigens used was a GST-H19G5 fusion protein containing amino acid residues 610 to 811 of SEQ ID NO: 1. Many clones of anti-19G5 monoclonal antibodies were obtained. Three of them were shown to recognize recombinant H19G5 proteins expressed in mammalian cells by Western analysis and immunocytochemistry. The antibodies may be used to determine the size and localization of 19G5 protein by Western blot and immunohistochemistry.

[0073] Subcellular localization of H19G5 protein was determined using confocal microscopy on cells transfected with vectors expressing 19G5 proteins fused to Green Fluorescent Protein (GFP). Three 19G5-GFP fusion protein expression constructs were made using three different 19G5 cDNA clones, the longest human 19G5 clone C11 [H19G5(C11)-GFP], a 2.7 kb clone of human 19G5 containing the C-terminal kinase domain [H19G59F1)-GFP], and the rat 19G5 small transcript [R19G5(S)-GFP]. The control GFP vector and the 19G5-GFP fusion expression constructs were transfected into C2C12 cells (ATCC Catalog No. CRL-1772). Cells were grown on chamber slides and transfected using SuperFectamine reagent from Quiagen for 3 hrs. Cells were fixed 24 hrs post-transfection with 4% paraformaldehyde for 15 min at room temperature and examined using a confocal microscope. The two longer forms of human 19G5-GFP proteins were detected in the nuclei whereas the short rat 19G5-GFP fusion protein was detected in the cytoplasm (FIG. 11). This suggests that different forms of 19G5 proteins are localized in different regions in the cells and may have different functions. Since the gene is normally expressed in heart, the localization of 19G5 protein in cardiac myocytes may be examined. Antisense constructs to study the effects of inhibiting the activity of 19G5 protein on cardiac myocytes may also be made and used.

[0074] A possibility that H19G5 might interact with various G proteins, as suggested by the presence of GEF domain and homology with Trio, was examined. HEK 293 cells constitutively expressing Epstein-Barr Virus Nuclear Antigen (EBNA) (Invitrogen, San Diego, Calif.) were transfected with H19G5-C11 expression construct. The eukaryotic vector used for 19G5 expression, pEAK8 (EdgeBiosystems), contains Epstein-Barr virus (EBV) origin of DNA replication, which allows replication of the expression construct in transfected cells thereby amplifying the level of protein expression. After 24 hrs, one 10 cm dish of transfected 293 EBNA cells were lysed with 1 ml of lysis buffer (1X PBS, 0.1% Triton, and proteinase inhibitors (0.2 mM AEBSF, 0.16 μM Aprotinin, 0.01 mM Bestatin, 3 μM E-64, 4 μM Leupeptin, and 2 μM Pepstatin) on ice for 30′. Cells were then homogenized with a dounce homogenizer on ice. The lysates were cleared by centrifugation. One ml of the cleared lysate was incubated with 5 μg of various small G proteins, expressed as GST fusion proteins and bound to glutathione-agarose beads, at 4° C. for 2 hrs. The protein-agarose complex was pelleted by brief centrifugation and washed for 4 times 5′ each with the lysis buffer at room temperature. The complex was then resuspended in SDS sample buffer, boiled and run on a SDS-PAGE and Western blotted with an anti-H19G5 monoclonal antibody. As shown in FIG. 12, H19G5 was found to bind to Cdc42. No binding was detected with Rac1 or RhoA. Lane 8 is His-tagged Cdc42 and in lane 9 His-tagged Cdc42-agarose complex was boiled for 5′ before adding to the H19G5 cell lysate. This result showed that H19G5 specifically interacts with Cdc42 protein and there is no non-specific interaction with agarose beads. The results presented herein suggest that 19G5 protein may play an important role, by virtue of regulating a small GTPase such as Cdc42Hs, in a variety of cellular activities. For example, Cdc42 has been shown to regulate actin polymerization and focal adhesion complex formation which in turn is necessary for filopedia formation (Nobes and Hall, Cell 81: 53-62[1995]). Cdc42 and rac have also been shown to regulate Jun N-terminal kinase (JNK) activity via the MAP kinase pathway (Coso et al., Cell 81: 1137-1146[1995]; Minden et al., Cell 81: 1147-1157[1995]; Olson et al., Science 269: 1270-1272 [1995]), an evolutionarily conserved and ubiquitous signal transduction pathway that impacts upon a number of important cellular functions.

[0075] C2C12 myoblasts cells (ATCC Catalog No. CRL-1772) can be induced to differentiate into myotubes when placed in a medium containing 2% horse serum (Lechner et al., Proc. Natl. Acad. Sci. USA 93: 4355-4359[1996]). FIG. 13 shows phase contrast micrographs of C2C12 myoblasts undergoing differentiation into myotubes. At Day 0, undifferentiated C2C12 cells with typical myoblast morphology can be seen. Once induced to differentiate, as shown here at Day 3 and 5 after induction, an increasingly larger number of cells with typical morphology of differentiated myotubes, i.e. large, elongated, multinucleated syncytial cells, could be seen. The induction of Myogenin expression was monitored during differentiation. Myogenin is not expressed in myoblasts, however, its expression is strongly induced when myoblasts undergo differentiation into myotubes. Thus, it acts as a biochemical marker of myotubes. FIG. 14 shows induction of myogenin protein expression when C2C12 myoblast cells are induced to differentiate into myotubes. C2C12 cells were cultured and induced to differentiate by placing in a medium containing 2% horse serum. Cells were lysed in radioimmunoprecipitation (RIPA) buffer (1X PBS containing 1% Igepal CA-630, 0.5% sodium deoxycholate and 0.1% SDS) on ice for 30′. Total lysates were cleared by centrifugation at 10,000 rpm for 10′. Protein concentration of each lysate was measure using the BCA method. SDS sample buffer was added to the total lysate and boiled for 3′. Equal amount of total protein of each sample was run on SDS-PAGE and blotted using an anti-myogenin monoclonal antibody. The result demonstrated that Myogenin expression was induced concomitantly with differentiation of C2C12 myoblasts into myotubes under the conditions used for induction.

[0076] The expression of 19G5 RNA was also monitored at various stages during differentiation of myoblasts into myotubes. Undifferentiated C2C12 cells were plated on 6 cm dishes at 2.4×10⁴ cells/cm² and cultured in growth medium with 10% fetal bovine serum for 24 hrs to about confluence. Cells were washed with PBS and induced to differentiate into myotubes in differentiation medium with 2% horse serum. Total RNA was isolated from cells at 1, 2, or 4 days post-induction using Qiagen's Rneasy kit according to the manufacture's instruction. The expression of 19G5 transcript was analyzed using Taqman assay. One microgram of total RNA, isolated at various time points post-induction, was reverse transcribed into cDNA using PE Biosystems Reagents and Multiscribe enzyme according to manufacture's instruction. Ten ng of cDNA was added to 1× master mix, and the primers and probe for the gene of interest were added according to manufacture's instructions. The reaction were carried out in the ABI Prism 7700 Detection System. The quantity of 19G5 and the quantity for 18S were determined for each sample, and the ratio of 19G5/18S was used to evaluate differences in the level of 19G5 expression in various samples. Comparison of the values thus obtained with pre-induction values allowed to determine fold induction of 19G5 expression during differentiation. As shown in FIG. 15, expression of 19G5 transcript was significantly increased during differentiation of C2C12 myoblasts into myotubes. The level of induction reached to about 10-fold at 4 days post-induction. Increased expression of 19G5 likely reflects a specific function in myotubes.

[0077] TGF-β is known to inhibit differentiation of C2C12 myoblast into myotubes (Katagiri et al., J. Cell Biol. 127: 1755-1766[1994]; Namiki et al., J. Biol. Chem. 272: 22046-22052[1997]). Therefore, the effect of TGF-β on the induction of expression of 19G5 during differentiation of C2C12 cells was examined. C2C12 cells were plated in 6 cm dishes at 2.4×10⁴ cells/cm² and cultured in the growth medium for 24 hours. Cells were then rinsed with PBS and induced to differentiate for 4 days in the medium containing 5% fetal bovine serum either in the absence or in the presence of 10 ng/ml of TGF-β. Total RNA was isolated from cells using Qiagen's Rneasy kit. Induction of 19G5 expression in TGF-β treated or untreated cells over undifferentiated C2C12 cells was measured by Taqman assay. As shown in FIG. 16, the induction of 19G5 expression during C2C12 differentiation is inhibited by TFG-β. TGF-β is a powerful regulator of cell growth and differentiation and regulation of expression of 19G5 by TGF-β likely represents an important physiological event with significant relevance to normal and abnormal changes in cardiac cells. Furthermore, the results suggest a possible inolvement of 19G5 in myogenesis.

EXAMPLE 2 Polypeptides which can Mediate Signal Transduction

[0078] The polypeptides of the present invention, such as the specific embodiment shown in SEQ ID NOs: 1, 4, 6, 7, 8 or 9 may be prepared by any known techniques. Conveniently, the polypeptides may be prepared using the solid-phase synthetic technique initially described by Merrifield in J. Am. Chem. Soc. 15:2149-2154 (1963). Other peptide synthesis techniques may be found, for example, in M. Bodanszky et al., PEPTIDE SYNTHESIS, John Wiley & Sons, 2d Ed. (1976) as well as in other reference works known to those skilled in the art. A summary of peptide synthesis techniques may be found in J. Stuart and J. D. Young, SOLID PHASE PEPTIDE SYNTHELIA, Pierce Chemical Co., Rockford, Ill. (1984). The synthesis of peptides by solution methods may also be used, as described in THE PROTEINS, Vol-II, 3d Ed., Neurath, H. et al., Eds., p.105-237, Academic Press, New York, N.Y. (1976). Appropriate protective groups for use in such syntheses will be found in the above texts as well as in J. F. W. McOmie, PROTECTIVE GROUPS IN ORGANIC CHEMISTRY, Plenum Press, New York, N.Y. (1973). In general, these synthetic methods involve the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively-removable protecting group. A different, selectively-removable protecting group is utilized for amino acids containing a reactive side group, such as lysine.

[0079] Using a solid phase synthesis as an example, the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group. The protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted under conditions suitable for forming the amide linkage with the residue already attached to the solid support. The protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to provide the final peptide. The polypeptides of the invention preferably are devoid of benzylated or methylbenzylated amino acids. Such protecting group moieties may be used in the course of synthesis, but they are removed before the polypeptides are used. Additional reactions may be necessary, as described elsewhere to form intramolecular linkages to restrain conformation, if desired. The polypeptides of the present invention may also be linked to an additional sequence of amino acids either or both at the N-terminus and at the C-terminus. Such additional amino acid sequences, or linker sequences, can be conveniently affixed to a detectable label, solid matrix, or carrier. Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic acid and aspartic acid, or the like.

[0080] Of course, the present polypeptides may also be prepared by recombinant DNA techniques as described, for example, in Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL, Chapters 1-18, Second Edition (Cold Spring Harbor N.Y. 1989), and as detailed in Examples 3-4 infra. The present invention also relates to vectors comprising DNA molecules of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques. Host cells may be genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying genes. The culture conditions, such as temperature, pH and the like, are preferably those previously used with the host cell selected for expression, and will be apparent to the skilled artisan.

EXAMPLE 3 Hosts, Vectors and DNA Encoding Polypeptides which can Mediate Signal Transduction

[0081] The DNA molecules of the present invention may be employed for producing the polypeptides of the present invention by recombinant techniques. More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences broadly described above. Thus, for example, the DNA molecule sequence may be included in any one of a variety of expression vehicles, in particular vectors or plasmids for expressing such a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA; viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTRC99A, pKK223-3, pKK233-3, pDR540, PRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, PSVL (Pharmacia). However, any other vector or plasmid may be used as long as they are replicable and viable in the host. The vector containing the appropriate DNA sequence, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the polypeptides of the present invention. Representative examples of appropriate hosts include: bacterial cells, such as E. coli, Salmonella typhimurium, Streptomyces; fungal cells, such as yeast; insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells, such as CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art.

[0082] The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

[0083] The DNA sequence in the expression vector may be operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. Suitable promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include laci, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of skill in the art. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

[0084] In addition, the expression vectors preferably may contain a gene to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

[0085] An embodiment of the invention is an isolated DNA molecule comprising the nucleotide sequence of SEQ ID NOs: 2, 3 or 5. This nucleotide sequence, or fragments or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of the polypeptides of the present invention, or functionally active peptides or functional equivalents thereof, in appropriate host cells. Due to the degeneracy of the nucleotide coding sequence, other DNA sequences which encode substantially the same amino acid sequences as depicted in SEQ ID NOS: 1, 4, 6, 7, 8 or 9, or analogs or fragments thereof, may be used in the practice of the invention for the cloning and expression of a mediator of signal transduction. Such alterations include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product. The gene product may contain deletions, additions or substitutions of amino acid residues within the sequence, which result in a silent change thus producing a bioactive product. Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, the amphipathic nature of the residues involved and/or on the basis of crystallographic data. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.

[0086] Techniques well known to those skilled in the art for the isolation of DNA, generation of appropriate restriction fragments, construction of clones and libraries, and screening recombinants may be used. For a review of such techniques, see, for example, Sambrook, et al., supra, the disclosure of which is hereby incorporated by reference. Also, the 5′ untranslated and coding regions of the nucleotide sequence could be altered to improve the translational efficiency of the mRNA. In addition, based on X-ray crystallographic data, sequence alterations could be undertaken to improve protein stability, e.g., introducing disulfide bridges at the appropriate positions, and/or deleting or replacing amino acids that are predicted to cause protein instability. These are only examples of modifications that can be engineered to produce a more active or stable protein, more protein, or even change the substrate specificity of the protein.

EXAMPLE 4 Cells Transformed with Recombinant Vectors Containing DNA Encoding Polypeptides which can Mediate Signal Transduction

[0087] In a further embodiment, the present invention relates to host cells containing the above-described construct. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. The host cell preferably may secrete the recombinant protein. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (L. Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, 1986)).

[0088] The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers. Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., supra.

[0089] Transcription of a DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin (base pair 100 to 270), a cytomegalovirus early promoter enhancer, a polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0090] Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), alpha factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is preferably assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

[0091] Useful expression vectors for bacterial use may be constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation, initiation and termination signals in operable reading phase with a functional promoter. The vector may comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

[0092] As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.). These pBR322 backbone sections are combined with an appropriate promoter and the structural sequence to be expressed.

[0093] Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter may be de-repressed by appropriate means (e.g., temperature shift or chemical induction) and cells may be cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

[0094] Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Various mammalian cell culture systems can also be employed to express recombinant polypeptides. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors may comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

[0095] The polypeptides of the present invention may be recovered and purified from recombinant cell cultures by methods used heretofore, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

[0096] The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic-procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

[0097] In particular, two baculovirus expression constructs of a wild-type and a mutant H19G5 C-terminal kinase domain have been constructed. The wild-type kinase domain construct was made by cloning a CDNA fragment which encodes the amino acid residues 1002 to 1314 of the H19G5 contig protein sequence into the pFastBac HTc vector. The mutant kinase domain construct contains the same amino acid sequence as the wild-type kinase domain except that Tyr residue at position 1213 was changed to a Glu in an attempt to create a constitutively active kinase. Both recombinant proteins contain a his-tag at the N-terminus.

EXAMPLE 5 Pharmaceutically Acceptable Salts of Polypeptides which can Mediate Signal Transduction

[0098] Any peptide of the present invention may be used in the form of a pharmaceutically acceptable salt. Suitable acids which are capable of forming salts with the peptides of the present invention include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid, and the like.

[0099] Suitable bases capable of forming salts with the peptides of the present invention include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine and the like).

EXAMPLE 6 Pharmaceutical Compositions Containing Polypeptides which can Mediate Signal Transduction

[0100] For use in a method of identification, prevention or treatment, such as the identification, prevention or treatment of infection of a mammalian host by a microorganism, the polypeptides of the present invention may be present in a pharmaceutical composition in admixture with a pharmaceutically acceptable sterile vehicle. The pharmaceutical composition may be compounded according to conventional pharmaceutical formulation techniques.

[0101] The vehicle may take a wide variety of forms depending on the form of preparation desired for administration, e.g., sublingual, rectal, nasal, oral or parenteral. Compositions for oral dosage form may include any of the usual pharmaceutical media, such as, for example, water, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations (e.g., suspensions, elixirs and solutions) or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (e.g., powders, capsules and tablets). Controlled release forms may also be used. Because of their ease in administration, tablets and capsules represent an advantageous oral dosage unit form, in which case solid pharmaceutical carriers may be employed. If desired, tablets may be sugar coated or enteric coated by standard techniques.

[0102] For compositions to be administered parenterally, the carrier will usually comprise sterile water, although other ingredients to aid solubility or for preservation purposes may be included. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. The parenteral routes of administration may be intravenous injection, intramuscular injection or subcutaneous injection.

[0103] For intravenous administration, the polypeptides may be dissolved in an appropriate intravenous delivery vehicle containing physiologically compatible substances such as sodium chloride, glycine and the like, having a buffered pH compatible with physiologic conditions. Such intravenous delivery vehicles are known to those skilled in the art.

[0104] The polypeptides of the invention may be administered to subjects where mediation of signal transduction is desired. The peptides may be administered by any convenient means that will result in the delivery to the subject of an effective amount to mediate signal transduction. Oral administration is presently contemplated as a preferred administration route. The amount administered will depend on the activity of the particular compound administered, which may readily be determined by those of ordinary skill in the art.

EXAMPLE 7 Monoclonal Antibodies Against Polypeptides which can Mediate Signal Transduction

[0105] Another embodiment of the present invention relates to a monoclonal antibody to the polypeptides of the present invention (or an antigenic portion thereof), which may be produced by methods recognized in the art, including the formation of monoclonal antibody-producing hybridomas (Kohler, G., and C. Milstein, Nature 256:495-497 (1975); Eur. J. Immunol. 6:511-519 (1976)). By fusing antibody-forming cells (spleen lymphocytes) with myeloma cells (malignant cells of bone marrow primary tumors), a hybrid cell line is created from a single fused cell hybrid (called a hybridoma or clone) having certain inherited characteristics of both the lymphocytes and myeloma cell lines. Like the lymphocytes (taken from animals primed with sheep red blood cells as antigen), the hybridomas secreted a single type of immunoglobulin specific to the antigen; moreover, like the myeloma cells, the hybrid cells had the potential for indefinite cell division. The combination of these two features offered distinct advantages over conventional antisera. Whereas antisera derived from vaccinated animals are variable mixtures of polyclonal antibodies which never can be reproduced identically, monoclonal antibodies are highly specific immunoglobulins of a single type. The single type of immunoglobulin secreted by a hybridoma is specific to one and only one antigenic determinant, or epitope, on the antigen, a complex molecule having a multiplicity of antigenic determinants. For instance, if the antigen is a protein, an antigenic determinant may be one of the many peptide sequences (generally 6-7 amino acids in length (Atassi, M. Z., Molec. Cell. Biochem. 32:21-43 (1980)) within the entire protein molecule. Hence, monoclonal antibodies raised against a single antigen may be distinct from each other depending on the determinant that induced their formation; but for any given clone, all of the antibodies it produces are identical. Furthermore, the hybridoma cell line can be reproduced indefinitely, is easily propagated in vitro or in vivo, and yields monoclonal antibodies in extremely high concentration.

EXAMPLE 8 Therapeutic Monoclonal Antibodies Against Polypeptides which can Mediate Signal Transduction

[0106] The monoclonal antibodies of the present invention can have potential immunotherapeutic value (Oldham, R. K., J. Clin. Oncol., 1:582-590 (1983); Miller, R. A. et al., Blood, 62:988-995 (1983); Miller R. A. et al., New Engl. J. Med. 306:517-522 (1982); Ritz, J. and Schlossman, S., Blood, 59:1-11 (1982); and Kirch, M. E. and Ulrich, H., J. Immunol. 127:805-810 (1981) (investigating the therapeutic efficacy in both animal and human subjects)). In addition, the monoclonal antibodies can be used in cytotoxic drug-antibody conjugates similar to those described in Beverly, P. C. L., Nature, 297:358-9 (1982); Krolick, K. A. et al., Nature, 295:604-5 (1982); Krolick, K. A. et al., Proc. Natl. Acad. Sci. U.S.A., 77:5419-23 (1980); Arnon, R. and Sela, M., Immunol. Rev., 62:5-27 (1982); Raso, V. et al., Cancer Res., 42:457-64 (1982); and DeWeger, R. A. and Dullens, H. F. J., Immunol. Rev. 62:29-45 (1982).

[0107] In an embodiment of the invention, purified polypeptides of the present invention (or an antigenic portion thereof) can be used as an antigen or immunogen. In addition, microorganisms expressing H19G5 protein or polypeptide fragments thereof also represent potential antigens or sources of antigen with which to immunize animals to obtain somatic cells for fusion. Somatic cells with the potential for producing antibody and, in particular, B lymphocytes, are suitable for fusion with a B-cell myeloma line. Those antibody-producing cells that are in the dividing plasmablast stage fuse preferentially. Somatic cells may be derived from the lymph nodes, spleens and peripheral blood of primed animals and the lymphatic cells of choice depending to a large extent on their empirical usefulness in the particular fusion system. Once-primed or hyperimmunized animals can be used as a source of antibody-producing lymphocytes. Mouse lymphocytes give a higher percentage of stable fusions with mouse myeloma lines. However, the use of rat, rabbit, and frog cells is also possible. Alternatively, human somatic cells capable of producing antibody, specifically B lymphocytes, are suitable for fusion with myeloma cell lines. While B lymphocytes from biopsied spleens or lymph nodes of individual may be used, the more easily accessible peripheral blood B lymphocytes are preferred. The lymphocytes may be derived from patients with diagnosed carcinomas.

[0108] Specialized myeloma cell lines have been developed from lymphocyte tumors for use in hybridoma-producing fusion procedures (Kohler, G., and C. Milstein, Eur. J. Immunol. 6:511-519 (1976); M. Schulman et al., Nature 276: 269-270 (1978)). Examples of mycloma cell lines that may be used for the production of fused cell hybrids include X63-Ag8, NSI-Ag4/1, MPC11-45.6TG1.7, C63-Ag8.653, Sp2/0-Ag14, FO, and S194/5XX0.BU.1, all derived from mice; 210.RCY3. Ag1.2.3, U-226AR, and GM1500GTGAL2, all derived from rats; and U-226AR and GM1500GTGAL2, derived from humans, (G. J. Hammerling, U. Hammerling, and J. F. Kearney (editors), Monoclonal Antibodies and T-cell Hybridomas in: J. L. Turk (editor) RESEARCH MONOGRAPHS IN IMMUNOLOGY, Vol. 3, Elsevier/North Holland Biomedical Press, NY (1981)).

[0109] Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion (though the proportion may vary from about 20:1 to about 1:1), respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. It is often preferred that the same species of animal serve as the source of the somatic and myeloma cells used in the fusion procedure. Fusion methods have been described by Kohler and Milstein (Nature 256:495-497 (1975) and Eur. J. Immunol. 6:511-519 (1976), and by Gefter et al. (Somatic Cell Genet. 3:231-236 (1977)). The fusion-promotion agents used by those investigators were Sendai virus and polyethylene glycol (PEG), respectively.

[0110] Once the desired fused cell hybrids have been selected and cloned into individual antibody-producing cell lines, each cell line may be propagated in either of two standard ways. A sample of the hybridoma can be injected into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can be tapped to provide monoclonal antibodies in high concentration. Alternatively, the individual cell lines may be propagated in vitro in laboratory culture vessels; the culture medium, also containing high concentrations of a single specific monoclonal antibody, can be harvested by decantation, filtration or centrifugation.

EXAMPLE 9 Diagnostic Monoclonal Antibodies Against Polypeptides which can Mediate Signal Transduction

[0111] The monoclonal antibodies of this invention can be used as probes in detecting discrete antigens expressed by tissue or cell samples. The expression or lack of expression of these antigens can provide clinically exploitable information that is not apparent after standard histopathological evaluations. It may thus be possible to correlate the immuno-phenotypes of individual tissue or cell samples with various aspects of signal transduction and responsiveness to certain types of therapies, thereby establishing important classifications of prognosis.

[0112] The use of the monoclonal antibodies described herein can be extended to the screening of human biological fluids for the presence of the specific antigenic determinant recognized. In vitro immunoserological evaluation of sera withdrawn from patients thereby permits non-invasive diagnosis of disease. By way of illustration, human fluids, such as pleural fluids or lymph, can be taken from a patient and assayed for the specific epitope, either as released antigen or membrane-bound on cells in the sample fluid, using monoclonal antibodies against the polypeptides of the present invention in standard radioimmunoassays or enzyme-linked immunoassays known in the art or competitive binding enzyme-linked immunoassays.

[0113] The monoclonal antibodies of this invention are potentially useful for targeting diseased tissue or cells in vivo. They can therefore be used in humans for localization and monitoring of the microbial infection. For this application, it is preferable to use purified monoclonal antibodies. Purification of monoclonal antibodies for human administration by ammonium sulfate or sodium sulfate precipitation followed by dialysis against saline and filtration sterilization has been described by Miller et al. (in: HYBRIDOMAS IN CANCER DIAGNOSIS AND THERAPY, (1982), p. 134).

[0114] Alternatively, immunoaffinity chromatography techniques may be used to purify the monoclonal antibodies. The purified monoclonal antibodies can be labeled with radioactive compounds, for instance, radioactive iodine, and administered to a patient intravenously. After localization of the antibodies at the infection site, they can be detected by emission tomographical and radionuclear scanning techniques, thereby pinpointing the location of the infection. Experimental radioimmunodetection with monoclonal antibodies may occur by external scintigraphy.

[0115] Passive monoclonal serotherapy may be a potential use for the monoclonal antibodies of this invention. By way of illustration, purified anti-H19G5 monoclonal antibody is dissolved in an appropriate carrier, e.g., saline, with or without human albumin, at an appropriate dosage and is administered to a patient. The monoclonal antibodies are preferably administered intravenously, e.g., by continuous intravenous infusion over several hours, as in Miller et al, supra. Infusions can be administered over a period of weeks during which the anti-microbial effects are monitored.

EXAMPLE 10 Anti-Idiotypic Antibodies to Antibodies Against Polypeptides which can Mediate Signal Transduction

[0116] In an alternate embodiment, the antibodies described herein are used to stimulate the production of corresponding anti-idiotypic antibodies. In brief, anti-idiotypic antibodies, or antiidiotypes are antibodies directed against the antigen combining region or variable region (idiotype) of another antibody. Based on Jerne's network model of idiotypic relationships (Jerne, Ann. Immunol. 125:373 (1974); Jerne et al., EMBO 1:234 (1982)), immunization with an antibody molecule expressing a paratope (antigen-combining site) for a given antigen should produce a group of anti-antibodies, some of which share with the antigen a complementary structure to the paratope. Immunization with a subpopulation of antiidiotypic antibodies should in turn produce a subpopulation of antiidiotypic antibodies which bind the initial antigen. Thus, the administration of the monoclonal antibodies of the present invention may result in a modification of the host's immune response, as the consequence of the formation of anti-idiotypic antibodies which may develop during therapy with the monoclonals.

EXAMPLE 11 Monoclonal Antibody-Drug Conjugates

[0117] The monoclonal antibodies of this invention can be used in conjunction with a broad spectrum of pharmaceutical or cytotoxic agents that selectively affect diseased tissue or cells over normal tissues or cells in the mammalian host. The methods used for binding the cytotoxic agents to the monoclonal antibody molecule can involve either non-covalent or covalent linkages. Since non-covalent bonds are more likely to be broken before the antibody complex reaches the target site, covalent linkages are preferred. For instance, carbodiimide can be used to link carboxy groups of the pharmaceutical agent to amino groups of the antibody molecule. Bifunctional agents such as dialdehydes or imidoesters can be used to link the amino group of a drug to amino groups of the antibody molecule. The Schiff base reaction can be used to link drugs to antibody molecules. This method involves the periodate oxidation of a drug or cytotoxic agent that contains a glycol or hydroxy group, thus forming an aldehyde that is then reacted with the antibody molecule. Attachment occurs via formation of a Schiff base with amino groups of the antibody molecule. Additionally, drugs with reactive sulfhydryl groups have been coupled to antibody molecules.

EXAMPLE 12 Diagnostic Kit

[0118] Another embodiment of the invention relates to a diagnostic kit for detecting diseased tissue or cells using an antibody against a polypeptide which can mediate signal transduction. The diagnostic kit may further comprise, where necessary, other components of the signal producing system, including agents for reducing background interference, control reagents, or an apparatus, container or other solid support for conducting the test. The binding of antibody to the target can be detected by well known methods, including radiation (e.g., use of a radioactive nucleotide), colorimetry (e.g., use of an enzyme that can cause a color change in a substrate), fluorescence (e.g., use of a dye such as propidium iodide, fluorescein, or rhodamine), and luminescence (e.g., use of an alkaline phosphatase substrate that releases photons upon cleavage or luciferin). Detection can be qualitative or quantitative.

EXAMPLE 13 Gene Therapy

[0119] Another embodiment of the present invention involves the use of the DNA of the present invention in gene therapy applications. Gene therapy has been broadly defined as “the correction of a disease phenotype through the introduction of new genetic information into the affected organism” (Roemer, K. and Friedmann, T., Eur. J. Biochem. 208: 211-225 (1992)). Two basic approaches to gene therapy have evolved: (1) ex vivo gene therapy and (2) in vivo gene therapy. In ex vivo gene therapy, cells are removed from a subject and cultured in vitro. A functional replacement gene is introduced into the cells (transfection) in vitro, the modified cells are expanded in culture, and then reimplanted in the subject. These genetically modified, reimplanted cells are reported to secrete detectable levels of the transfected gene product in situ (Miller, A. D., Blood 76: 271-278 (1990)) and Selden, R. F., et al., New Eng. J. Med. 317: 1067-1076 (1987)). The development of improved retroviral gene transfer methods (transduction) facilitates the transfer into and subsequent expression of genetic material by somatic cells (Cepko, C. L., et al., Cell 37: 1053-1062 (1984)). Accordingly, retrovirus-mediated gene transfer has been used in clinical trials to mark autologous cells and as a way of treating genetic disease (Rosenberg, S. A., et al., New Eng. J. Med. 323: 570-578 (1990); Anderson, W. F., Human Gene Therapy 2: 99-100 (1991)). Several ex vivo gene therapy studies in humans are reported (reviewed in Anderson, W. F., Science 256: 808-813 (1992) and Miller A. D., Nature 357: 455-460 (1992)).

[0120] In in vivo gene therapy, target cells are not removed from the subject. Rather, the transferred gene is introduced into cells of the recipient organism in situ, that is, within the recipient. In vivo gene therapy has been examined in several animal models (reviewed in Felgner, P. L. and Rhodes, G., Nature 349: 351-352 (1991)). Publications have reported the feasibility of direct gene transfer in situ into organs and tissues such as muscle (Ferry, N., et al., Proc. Natl. Acad. Sci. 88: 8377-8781 (1991); Quantin, G., et al., Proc. Natl. Acad. Sci. USA 89: 2581-2584 (1992)), hematopoietic stem cells (Clapp, D. W., et al., Blood 78: 1132-1139 (1991)), the arterial wall (Nabel, E. G., et al., Science 244: 1342-1344 (1989)), the nervous system (Price, J. D., et al., Proc. Natl. Acad. Sci. 84: 156-160 (1987)), and lung (Rosenfeld, M. A., et al., Science 252: 431-434 (1991)). Direct injection of DNA into skeletal muscle (Wolff, J. A., et al., Science 247: 1465-1468 (1990)), heart muscle (Kitsis, R. N., et al., Proc. Natl. Acad. Sci. USA 88: 4138-4142 (1991)) and injection of DNA-lipid complexes into the vasculature (Lim, C. S., et al., Circulation 83: 2007-2011 (1991); Ledere, G. D., et al., J. Clin. Invest. 90: 936-944 (1992); Chapman, G. D., et al., Circ. Res. 71: 27-33 (1992)) also have been reported to yield a detectable expression level of the inserted gene product(s) in vivo.

[0121] Recent gene therapy efforts have been aimed at the identification of various cell types for transformation, including keratinocytes (Morgan, J. R., et al., Science 237: 1476-1479 (1987)), fibroblasts (Palmer, T. D., et al., Proc. Natl. Acad. Sci. 88: 1330-1334 (1991); Garver Jr., R. I., et al., Science 237: 762-764 (1987); International Patent Application PCT/US92/01890, having publication number WO 92/15676), lymphocytes (Reimann, J. K., et al., J. Immunol. Methods 89: 93-101 (1986)), myoblasts (Barr, E. and Leiden, J. M., Science 254: 1507-1509 (1991); Dai, Y. et al., PNAS 89: 10892-10895 (1992); Roman, M., et al., Somatic Cell and Molecular Genetics 18: 247-258 (1992)), smooth muscle cells (Lynch, C. M. et al., Proc. Natl. Acad. Sci. USA 89: 1138-1142 (1992)), and epithelial cells (Nabel, E. G., et al., Science 244: 1342-1344 (1989)), International Patent Application PCT/US89/05575 (having publication number WO 90/06997), the contents of which references and patent/patent applications are incorporated herein by reference.

[0122] The delivery of an effective dose of a prophylactic or therapeutic agent in situ depends on the efficiency of transfection (or transduction) as well as the number of target cells. Epithelial cell-based gene therapy, in particular, involves a relatively small area available in situ for receiving genetically modified epithelial cells. The delivery of an effective dose of prophylactic or therapeutic agent in situ thus depends upon the total number of implanted epithelial cells.

[0123] In one embodiment of the invention, exogenous genetic material (e.g., a cDNA encoding a polypeptide of the present invention) is introduced into a syngeneic host cell ex vivo or in vivo by genetic transfer methods, such as transfection or transduction, to provide a genetically modified host cell. Various expression vectors (i.e., vehicles for facilitating delivery of exogenous genetic material into a target cell) are known to one skilled in the art.

[0124] Transfection refers to the insertion of nucleic acid into a mammalian host cell using physical or chemical methods. Several transfection techniques are known to those of ordinary skill in the art including: calcium phosphate DNA co-precipitation (METHODS IN MOLECULAR BIOLOGY, Vol. 7, Gene Transfer and Expression Protocols, Ed. E. J. Murray, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; and tungsten particle-facilitated microparticle bombardment (Johnston, S. A., Nature 346: 776-777 (1990)). Strontium phosphate DNA co-precipitation (Brash D. E. et al. Molec. Cell. Biol. 7: 2031-2034 (1987)) is a preferred transfection method.

[0125] In contrast, transduction refers to the process of transferring nucleic acid into a cell using a DNA or RNA virus. A RNA virus (i.e., a retrovirus) for transferring a nucleic acid into a cell is referred to herein as a transducing chimeric retrovirus. Exogenous genetic material contained within the retrovirus is incorporated into the genome of the transduced host cell. A host cell that has been transduced with a chimeric DNA virus (e.g., an adenovirus carrying a cDNA encoding a therapeutic agent) will not have the exogenous genetic material incorporated into its genome, but will be capable of expressing the exogenous genetic material that is retained extrachromosomally within the cell.

[0126] Typically, the exogenous genetic material includes the heterologous gene (usually in the form of a cDNA comprising the exons coding for the therapeutic protein) together with a promoter to control transcription of the new gene. The promoter characteristically has a specific nucleotide sequence necessary to initiate transcription. Optionally, the exogenous genetic material further includes additional sequences (i.e., enhancers) required to obtain the desired gene transcription activity. For the purpose of this discussion an enhancer is simply any non-translated DNA sequence which works contiguous with the coding sequence (in cis) to change the basal transcription level dictated by the promoter. Preferably, the exogenous genetic material is introduced into the host cell genome immediately downstream from the promoter so that the promoter and coding sequence are operatively linked so as to permit transcription of the coding sequence. A preferred retroviral expression vector includes an exogenous promoter element to control transcription of the inserted exogenous gene. Such exogenous promoters include both constitutive and inducible promoters.

[0127] Naturally-occurring constitutive promoters control the expression of essential cell functions. As a result, a gene under the control of a constitutive promoter is expressed under all conditions of cell growth. Exemplary constitutive promoters include the promoters for the following genes which encode certain constitutive or housekeeping functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR) (Scharfinann et al., Proc. Natl. Acad. Sci. USA 88: 4626-4630 (1991)), adenosine deaminase, phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, the beta-actin promoter (Lai et al., Proc. Natl. Acad. Sci. USA 86: 10006-10010 (1989)), and other constitutive promoters known to those of skill in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include: the early and late promoters of SV40, the long terminal repeats (LTRs) of Moloney Leukemia Virus and other retroviruses, and the thymidine kinase promoter of Herpes Simplex Virus, among many others. Accordingly, any such constitutive promoters can be used to control transcription of a heterologous gene insert.

[0128] Genes that are under the control of inducible promoters are expressed only or to a greater degree, in the presence of an inducing agent, (e.g., transcription under control of the metallothionein promoter is greatly increased in presence of certain metal ions). Inducible promoters include responsive elements (REs) which stimulate transcription when their inducing factors are bound. For example, there are REs for serum factors, steroid hormones, retinoic acid and cyclic AMP. Promoters containing a particular RE can be chosen in order to obtain an inducible response, and in some cases, the RE itself may be attached to a different promoter, thereby conferring inducibility to the recombinant gene. Thus, by selecting the appropriate promoter (constitutive versus inducible; strong versus weak), it is possible to control both the existence and level of expression of a therapeutic agent in the genetically modified host cell. If the gene encoding the prophylactic or therapeutic agent is under the control of an inducible promoter, delivery of the agent in situ is triggered by exposing the genetically modified cell in situ to conditions for permitting transcription of the prophylactic or therapeutic agent, e.g., by intraperitoneal injection of specific inducers of the inducible promoters which control transcription of the agent. For example, in situ expression by genetically modified host cells of a therapeutic agent encoded by a gene under the control of the metallothionein promoter, is enhanced by contacting the genetically modified cells with a solution containing the appropriate (i.e., inducing) metal ions in situ.

[0129] Accordingly, the amount of therapeutic agent that is delivered in situ is regulated by controlling such factors as: (1) the nature of the promoter used to direct transcription of the inserted gene (i.e., whether the promoter is constitutive or inducible, strong or weak); (2) the number of copies of the exogenous gene that are inserted into the host cell; (3) the number of transduced/transfected host cells that are administered (e.g., implanted) to the patient; (4) the size of the implant (e.g., graft or encapsulated expression system); (5) the number of implants; (6) the length of time the transduced/transfected cells or implants are left in place; and (7) the production rate of the prophylactic or therapeutic agent by the genetically modified host cell. Selection and optimization of these factors for delivery of an effective dose of a particular prophylactic or therapeutic agent is deemed to be within the scope of one of skill in the art, taking into account the above-disclosed factors and the clinical profile of the patient.

[0130] In addition to at least one promoter and at least one heterologous nucleic acid encoding the prophylactic or therapeutic agent, the expression vector preferably includes a selection gene, for example, a neomycin resistance gene, for facilitating selection of host cells that have been transfected or transduced with the expression vector. Alternatively, the host cells are transfected with two or more expression vectors, at least one vector containing the gene(s) encoding the prophylactic or therapeutic agent(s), the other vector containing a selection gene. The selection of a suitable promoter, enhancer, selection gene and/or signal sequence is deemed to be within the scope of one skilled in the art.

[0131] The prophylactic or therapeutic agent can be targeted for delivery to an extracellular, intracellular or membrane location. If it is desirable for the gene product to be secreted from the host cells, the expression vector is designed to include an appropriate secretion signal sequence for secreting the therapeutic gene product from the cell to the extracellular milieu. If it is desirable for the gene product to be retained within the host cell, this secretion signal sequence is omitted. In a similar manner, the expression vector can be constructed to include retention signal sequences for anchoring the prophylactic or therapeutic agent within the host cell plasma membrane. For example, membrane proteins have hydrophobic transmembrane regions that stop translocation of the protein in the membrane and do not allow the protein to be secreted. The construction of an expression vector including signal sequences for targeting a gene product to a particular location is deemed to be within the scope of one of skill in the art.

[0132] In an embodiment, vectors for mammalian host cell gene therapy are viruses, more preferably replication-deficient viruses (described in detail below). Exemplary viral vectors are derived from: Harvey Sarcoma Virus; Rous Sarcoma Virus, MPSV, Moloney murine leukemia virus and DNA viruses (e.g., adenovirus). See Temin, H., Retrovirus vectors for gene transfer, in GENE TRANSFER, Kucherlapati R, Ed., pp. 149-187 (Plenum 1986).

[0133] Replication-deficient retroviruses are capable of directing synthesis of virion proteins, but are incapable of making infectious particles. Accordingly, these genetically altered retroviral expression vectors have general utility for high-efficiency transduction of genes in cultured cells, and specific utility for use in the method of the present invention. Such retroviruses further have utility for the efficient transduction of genes into host cells in vivo. Retroviruses have been used extensively for transferring genetic material into cells. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell line with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with the viral particles) are provided in Kriegler, M. GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, W. H. Freeman Co., NY (1990) and Murray, E. J., ed. METHODS IN MOLECULAR BIOLOGY, Vol. 7, Humana Press Inc., Clifton, N.J. (1991).

[0134] The major advantage of using retroviruses for gene therapy is that the viruses insert the gene encoding the therapeutic agent into the host cell genome, thereby permitting the exogenous genetic material to be passed on to the progeny of the cell when it divides. In addition, gene promoter sequences in the LTR region have been reported to enhance expression of an inserted coding sequence in a variety of cell types (see e.g., Hilberg et al., Proc. Natl. Acad. Sci. USA 84: 5232-5236 (1987); Holland et al., Proc. Natl. Acad. Sci. USA 84: 8662-8666 (1987); Valerio et al., Gene 84: 419-427 (1989)). In vivo gene therapy using replication-deficient retroviral vectors to deliver a therapeutically effective amount of a therapeutic agent can be efficacious if the efficiency of transduction is high and/or the number of target cells available for transduction is high.

[0135] Yet another viral candidate useful as an expression vector for transformation of mammalian host cells is the adenovirus, a double-stranded DNA virus. The adenovirus is frequently responsible for respiratory tract infections in humans and thus appears to have an avidity for the epithelium of the respiratory tract (Straus, S., THE ADENOVIRUS, H. S. Ginsberg, Editor, Plenum Press, NY, p.451-496 (1984)). Moreover, the adenovirus is infective in a wide range of cell types, including, for example, muscle and epithelial cells (Larrick, J. W. and Burck, K. L., GENE THERAPY. APPLICATION OF MOLECULAR BIOLOGY, Elsevier Science Publishing Co., Inc., NY, p.71-104 (1991)). The adenovirus also has been used as an expression vector in muscle cells in vivo (Quantin, B., et al., Proc. Natl. Acad. Sci. USA 89: 2581-2584 (1992)).

[0136] Like the retrovirus, the adenovirus genome is adaptable for use as an expression vector for gene therapy, i.e., by removing the genetic information that controls production of the virus itself (Rosenfeld, M. A., et al., Science 252:431-434 (1991)). Because the adenovirus functions in an extrachromosomal fashion, the recombinant adenovirus does not have the theoretical problem of insertional mutagenesis.

[0137] Thus, as will be apparent to one skilled in the art, a variety of suitable viral expression vectors are available for transferring exogenous genetic material into mammalian host cells. The selection of an appropriate expression vector to express an agent for the identification, prevention or treatment of microbial infection amenable to gene replacement therapy and the optimization of the conditions for insertion of the selected expression vector into the cell are within the scope of one of skill in the art without the need for undue experimentation.

[0138] In an alternative embodiment, the expression vector is in the form of a plasmid, which is transferred into the target host cells by one of a variety of methods: physical (e.g., microinjection (Capecchi, M. R., Cell 22: 479-488 (1980)), electroporation (Andreason, G. L. and Evans, G. A. Biotechniques 6: 650-660 (1988)), scrape loading, microparticle bombardment (Johnston, S. A., Nature 346: 776-777 (1990)) or by cellular uptake as a chemical complex (e.g., calcium or strontium co-precipitation, complexation with lipid, complexation with ligand) (METHODS IN MOLECULAR BIOLOGY, Vol. 7, GENE TRANSFER AND EXPRESSION PROTOCOLS, Ed. E. J. Murray, Humana Press (1991)). Several commercial products are available for cationic liposome complexation including Lipofectin (Life Technologies, Inc., Gaithersburg, Md.) (Felgner, P. L., et al., Proc. Natl. Acad. Sci. 84: 7413-7417 (1987)) and Transfectam™ (ProMega, Madison, Wis.) (Behr, J. P., et al., Proc. Natl. Acad. Sci. USA 86: 6982-6986 (1989); Loeffler, J. P., et al., J. Neurochem. 54: 1812-1815 (1990)). However, the efficiency of transfection by these methods is highly dependent on the nature of the target cell and accordingly, the conditions for optimal transfection of nucleic acids into host cells using the above-mentioned procedures must be optimized. Such optimization is within the scope of one of skill in the art.

[0139] In an embodiment, the preparation of genetically modified host cells contains an amount of cells sufficient to deliver a prophylactically or therapeutically effective dose of a signal transduction mediator of the present invention to the recipient in situ. The determination of an effective dose of the prophylactic or therapeutic agent for a known microbial infection is within the scope of one of skill in the art. Thus, in determining the effective dose, the skilled artisan would consider the condition of the patient, the severity of the condition, as well as the results of clinical studies of the prophylactic or therapeutic agent being administered.

[0140] If the genetically modified host cells are not already present in a pharmaceutically acceptable carrier, they are placed in such a carrier prior to administration to the recipient. Such pharmaceutically acceptable carriers include, for example, isotonic saline and other buffers as appropriate to the patient and therapy. The genetically modified cells are administered by, for example, intraperitoneal injecting or implanting the cells or a graft or capsule containing the cells in a host cell-compatible site of the recipient. As used herein, host cell-compatible site refers to a structure, cavity or fluid of the recipient into which the genetically modified cell(s), host cell graft, or encapsulated host cell expression system can be implanted, without triggering adverse physiological consequences. Representative host cell-compatible sites include, for example, the peritoneal, pleural and pericardial cavities. Preferably, the host cell-compatible site communicates with the lymphatic system, thereby enabling delivery of the therapeutic agent to the vascular system.

[0141] In one embodiment, the host cell-compatible site may be denuded prior to implanting the cells. Exemplary denuding methods include but are not limited to: (1) injection of distilled water into the site (e.g., the peritoneal cavity) for 20 minutes, followed by scraping off a portion of the epithelial layer; (2) injection of 0.1% buffered trypsin for 20 minutes followed by scraping; (3) removal of epithelial cells by gentle scraping with a cell scraper and (4) touching a piece of Gelfilm (Upjohn, Kalamazoo, Mich.) to the endothelium.

[0142] The genetically modified host cells are implanted in a host cell-compatible site, alone or in combination with other genetically modified host cells. Thus, the instant invention embraces a method for modifying the epithelial system of a recipient by using a mixture of genetically modified host cells, such that a first modified cell expresses a first prophylactic or therapeutic agent of the present invention and a second modified cell expresses a second prophylactic or therapeutic agent. Other genetically modified cell types (e.g., hepatocytes, smooth muscle cells, fibroblasts, glial cells, mesothelial cells or keratinocytes) can be added, together with the genetically altered epithelial cells, to produce expression of a complex set of introduced genes. Moreover, more than one recombinant gene can be introduced into each genetically modified cell on the same or different vectors, thereby allowing the expression of multiple prophylactic or therapeutic agents of the present invention by a single cell.

[0143] The instant invention further embraces an epithelial cell graft. The graft comprises a plurality of the above-described genetically modified cells attached to a support that is suitable for implantation into a mammalian recipient, preferably into the oral cavity. The support can be formed of a natural or synthetic material. According to another aspect of the invention, an encapsulated host cell expression system is provided. The encapsulated system includes a capsule suitable for implantation into a mammalian recipient and a plurality of the above-described genetically modified host cells contained therein. The capsule can be formed of a synthetic or naturally-occurring material. The formulation of such capsules is known to one of ordinary skill in the art. In contrast to the host cells that are directly implanted into the mammalian recipient (i.e., implanted in a manner such that the genetically modified cells are in direct physical contact with the host cell-compatible site), the encapsulated cells remain isolated (i.e., not in direct physical contact with the site) following implantation. Thus, the encapsulated host cell system is not limited to a capsule including genetically-modified non-immortalized host cells, but may contain genetically modified immortalized host cells.

[0144] The invention has been disclosed broadly and illustrated in reference to representative embodiments described above. Those skilled in the art will recognize that various modifications can be made to the present invention without departing from the spirit and scope thereof.

1 9 1 1351 PRT Homo sapiens 1 Gly Val Gln Phe Ser Gln Tyr Gly Ser Pro Glu Phe Val Ser Pro Glu 1 5 10 15 Ile Ile Gln Gln Asn Pro Val Ser Glu Ala Ser Asp Ile Trp Ala Met 20 25 30 Gly Val Ile Ser Tyr Leu Ser Leu Thr Cys Ser Ser Pro Phe Ala Gly 35 40 45 Glu Ser Asp Arg Ala Thr Leu Leu Asn Val Leu Glu Gly Arg Val Ser 50 55 60 Trp Ser Ser Pro Met Ala Ala His Leu Ser Glu Asp Ala Lys Asp Phe 65 70 75 80 Ile Lys Ala Thr Leu Gln Arg Ala Pro Gln Ala Arg Pro Ser Ala Ala 85 90 95 Gln Cys Leu Ser His Pro Trp Phe Leu Lys Ser Met Pro Ala Glu Glu 100 105 110 Ala His Phe Ile Asn Thr Lys Gln Leu Lys Phe Leu Leu Ala Arg Ser 115 120 125 Arg Trp Gln Arg Ser Leu Met Ser Tyr Lys Ser Ile Leu Val Met Arg 130 135 140 Ser Ile Pro Glu Leu Leu Arg Gly Pro Pro Asp Ser Pro Ser Leu Gly 145 150 155 160 Val Ala Arg His Leu Cys Arg Asp Thr Gly Gly Ser Ser Ser Ser Ser 165 170 175 Ser Ser Ser Asp Asn Glu Leu Ala Pro Phe Ala Arg Ala Lys Ser Leu 180 185 190 Pro Pro Ser Pro Val Thr His Ser Pro Leu Leu His Pro Arg Gly Phe 195 200 205 Leu Arg Pro Ser Ala Ser Leu Pro Glu Glu Ala Glu Ala Ser Glu Arg 210 215 220 Ser Thr Glu Ala Pro Ala Pro Pro Ala Ser Pro Glu Gly Ala Gly Pro 225 230 235 240 Pro Ala Ala Gln Gly Cys Val Pro Arg His Ser Val Ile Arg Ser Leu 245 250 255 Phe Tyr His Gln Ala Gly Glu Ser Pro Glu His Gly Ala Leu Ala Pro 260 265 270 Gly Ser Arg Arg His Pro Ala Arg Arg Arg His Leu Leu Lys Gly Gly 275 280 285 Tyr Ile Ala Gly Ala Leu Pro Gly Leu Arg Glu Pro Leu Met Glu His 290 295 300 Arg Val Leu Glu Glu Glu Ala Ala Arg Glu Glu Gln Ala Thr Leu Leu 305 310 315 320 Ala Lys Ala Pro Ser Phe Glu Thr Ala Leu Arg Leu Pro Ala Ser Gly 325 330 335 Thr His Leu Ala Pro Gly His Ser His Ser Leu Glu His Asp Ser Pro 340 345 350 Ser Thr Pro Arg Pro Ser Ser Glu Ala Cys Gly Glu Ala Gln Arg Leu 355 360 365 Pro Ser Ala Pro Ser Gly Gly Ala Pro Ile Arg Asp Met Gly His Pro 370 375 380 Gln Gly Ser Lys Gln Leu Pro Ser Thr Gly Gly His Pro Gly Thr Ala 385 390 395 400 Gln Pro Glu Arg Pro Ser Pro Asp Ser Pro Trp Gly Gln Pro Ala Pro 405 410 415 Phe Cys His Pro Lys Gln Gly Ser Ala Pro Gln Glu Gly Cys Ser Pro 420 425 430 His Pro Ala Val Ala Pro Cys Pro Pro Gly Ser Phe Pro Pro Gly Ser 435 440 445 Cys Lys Glu Ala Pro Leu Val Pro Ser Ser Pro Phe Leu Gly Gln Pro 450 455 460 Gln Ala Pro Pro Ala Pro Ala Lys Ala Ser Pro Pro Leu Asp Ser Lys 465 470 475 480 Met Gly Pro Gly Asp Ile Ser Leu Pro Gly Arg Pro Lys Pro Gly Pro 485 490 495 Cys Ser Ser Pro Gly Ser Ala Ser Gln Ala Ser Ser Ser Gln Val Ser 500 505 510 Ser Leu Arg Val Gly Ser Ser Gln Val Gly Thr Glu Pro Gly Pro Ser 515 520 525 Leu Asp Ala Glu Gly Trp Thr Gln Glu Ala Glu Asp Leu Ser Asp Ser 530 535 540 Thr Pro Thr Leu Gln Arg Pro Gln Glu Gln Ala Thr Met Arg Lys Phe 545 550 555 560 Ser Leu Gly Gly Arg Gly Gly Tyr Ala Gly Val Ala Gly Tyr Gly Thr 565 570 575 Phe Ala Phe Gly Gly Asp Ala Gly Gly Met Leu Gly Gln Gly Pro Met 580 585 590 Trp Ala Arg Ile Ala Trp Ala Val Ser Gln Ser Glu Glu Glu Glu Gln 595 600 605 Glu Glu Ala Arg Ala Glu Ser Gln Ser Glu Glu Gln Gln Glu Ala Arg 610 615 620 Ala Glu Ser Pro Leu Pro Gln Val Ser Ala Arg Pro Val Pro Glu Val 625 630 635 640 Gly Arg Ala Pro Thr Arg Ser Ser Pro Glu Pro Thr Pro Trp Glu Asp 645 650 655 Ile Gly Gln Val Ser Leu Val Gln Ile Arg Asp Leu Ser Gly Asp Ala 660 665 670 Glu Ala Ala Asp Thr Ile Ser Leu Asp Ile Ser Glu Val Asp Pro Ala 675 680 685 Tyr Leu Asn Leu Ser Asp Leu Tyr Asp Ile Lys Tyr Leu Pro Phe Glu 690 695 700 Phe Met Ile Phe Arg Lys Val Pro Lys Ser Ala Gln Pro Glu Pro Pro 705 710 715 720 Ser Pro Met Ala Glu Glu Glu Leu Ala Glu Phe Pro Glu Pro Thr Trp 725 730 735 Pro Trp Pro Gly Glu Leu Gly Pro His Ala Gly Leu Glu Ile Thr Glu 740 745 750 Glu Ser Glu Asp Val Asp Ala Leu Leu Ala Glu Ala Ala Val Gly Arg 755 760 765 Lys Arg Lys Trp Ser Ser Pro Ser Arg Ser Leu Phe His Phe Pro Gly 770 775 780 Arg His Leu Pro Leu Asp Glu Pro Ala Glu Leu Gly Leu Arg Glu Arg 785 790 795 800 Val Lys Ala Ser Val Glu His Ile Ser Arg Ile Leu Lys Gly Arg Pro 805 810 815 Glu Gly Leu Glu Lys Glu Gly Pro Pro Arg Lys Lys Pro Gly Leu Ala 820 825 830 Ser Phe Arg Leu Ser Gly Leu Lys Ser Trp Asp Arg Ala Pro Thr Phe 835 840 845 Leu Arg Glu Leu Ser Asp Glu Thr Val Val Leu Gly Gln Ser Val Thr 850 855 860 Leu Ala Cys Gln Val Ser Ala Gln Pro Ala Ala Gln Ala Thr Trp Ser 865 870 875 880 Lys Asp Gly Ala Pro Leu Glu Ser Ser Ser Arg Val Leu Ile Ser Ala 885 890 895 Thr Leu Lys Asn Phe Gln Leu Leu Thr Ile Leu Val Val Val Ala Glu 900 905 910 Asp Leu Gly Val Tyr Thr Cys Ser Val Ser Asn Ala Leu Gly Thr Val 915 920 925 Thr Thr Thr Gly Val Leu Arg Lys Ala Glu Arg Pro Ser Ser Ser Pro 930 935 940 Cys Pro Asp Ile Gly Glu Val Tyr Ala Asp Gly Val Leu Leu Val Trp 945 950 955 960 Lys Pro Val Glu Ser Tyr Gly Pro Val Thr Tyr Ile Val Gln Cys Ser 965 970 975 Leu Glu Gly Gly Ser Trp Thr Thr Leu Ala Ser Asp Ile Phe Asp Cys 980 985 990 Cys Tyr Leu Thr Ser Lys Leu Ser Arg Gly Gly Thr Tyr Thr Phe Arg 995 1000 1005 Thr Ala Cys Val Ser Lys Ala Gly Met Gly Pro Tyr Ser Ser Pro Ser 1010 1015 1020 Glu Gln Val Leu Leu Gly Gly Pro Ser His Leu Ala Ser Glu Glu Glu 1025 1030 1035 1040 Ser Gln Gly Arg Ser Ala Gln Pro Leu Pro Ser Thr Lys Thr Phe Ala 1045 1050 1055 Phe Gln Thr Gln Ile Gln Arg Gly Arg Phe Ser Val Val Arg Gln Cys 1060 1065 1070 Trp Glu Lys Ala Ser Gly Arg Ala Leu Ala Ala Lys Ile Ile Pro Tyr 1075 1080 1085 His Pro Lys Asp Lys Thr Ala Val Leu Arg Glu Tyr Glu Ala Leu Lys 1090 1095 1100 Gly Leu Arg His Pro His Leu Ala Gln Leu His Ala Ala Tyr Leu Ser 1105 1110 1115 1120 Pro Arg His Leu Val Leu Ile Leu Glu Leu Cys Ser Gly Pro Glu Leu 1125 1130 1135 Leu Pro Cys Leu Ala Glu Arg Ala Ser Tyr Ser Glu Ser Glu Val Lys 1140 1145 1150 Asp Tyr Leu Trp Gln Met Leu Ser Ala Thr Gln Tyr Leu His Asn Gln 1155 1160 1165 His Ile Leu His Leu Asp Leu Arg Ser Glu Asn Met Ile Ile Thr Glu 1170 1175 1180 Tyr Asn Leu Leu Lys Val Val Asp Leu Gly Asn Ala Gln Ser Leu Ser 1185 1190 1195 1200 Gln Glu Lys Val Leu Pro Ser Asp Lys Phe Lys Asp Tyr Leu Glu Thr 1205 1210 1215 Met Ala Pro Glu Leu Leu Glu Gly Gln Gly Ala Val Pro Gln Thr Asp 1220 1225 1230 Ile Trp Ala Ile Gly Val Thr Ala Phe Ile Met Leu Ser Ala Glu Tyr 1235 1240 1245 Pro Val Ser Ser Glu Gly Ala Arg Asp Leu Gln Arg Gly Leu Arg Lys 1250 1255 1260 Gly Leu Val Arg Leu Ser Arg Cys Tyr Ala Gly Leu Ser Gly Gly Ala 1265 1270 1275 1280 Val Ala Phe Leu Arg Ser Thr Leu Cys Ala Gln Pro Trp Gly Arg Pro 1285 1290 1295 Cys Ala Ser Ser Cys Leu Gln Cys Pro Trp Leu Thr Glu Glu Gly Pro 1300 1305 1310 Ala Cys Ser Arg Pro Ala Pro Val Thr Phe Pro Thr Ala Arg Leu Arg 1315 1320 1325 Val Phe Val Arg Asn Arg Glu Lys Arg Arg Ala Leu Leu Tyr Lys Arg 1330 1335 1340 His Asn Leu Ala Gln Val Arg 1345 1350 2 4175 DNA Homo sapiens CDS (1)...(4053) 2 gga gtg cag ttc agc cag tac ggc tcc cct gag ttc gtc tcc ccc gag 48 Gly Val Gln Phe Ser Gln Tyr Gly Ser Pro Glu Phe Val Ser Pro Glu 1 5 10 15 atc atc cag cag aac cct gtg agc gaa gcc tcc gac att tgg gcc atg 96 Ile Ile Gln Gln Asn Pro Val Ser Glu Ala Ser Asp Ile Trp Ala Met 20 25 30 ggt gtc atc tcc tac ctc agc ctg acc tgc tca tcc cca ttt gcc ggc 144 Gly Val Ile Ser Tyr Leu Ser Leu Thr Cys Ser Ser Pro Phe Ala Gly 35 40 45 gag agt gac cgt gcc acc ctc ctg aac gtc ctg gag ggg cgc gtg tca 192 Glu Ser Asp Arg Ala Thr Leu Leu Asn Val Leu Glu Gly Arg Val Ser 50 55 60 tgg agc agc ccc atg gct gcc cac ctc agc gaa gac gcc aaa gac ttc 240 Trp Ser Ser Pro Met Ala Ala His Leu Ser Glu Asp Ala Lys Asp Phe 65 70 75 80 atc aag gct acg ctg cag aga gcc cct cag gcc cgg cct agt gcg gcc 288 Ile Lys Ala Thr Leu Gln Arg Ala Pro Gln Ala Arg Pro Ser Ala Ala 85 90 95 cag tgc ctc tcc cac ccc tgg ttc ctg aaa tcc atg cct gcg gag gag 336 Gln Cys Leu Ser His Pro Trp Phe Leu Lys Ser Met Pro Ala Glu Glu 100 105 110 gcc cac ttc atc aac acc aag cag ctc aag ttc ctc ctg gcc cga agt 384 Ala His Phe Ile Asn Thr Lys Gln Leu Lys Phe Leu Leu Ala Arg Ser 115 120 125 cgc tgg cag cgt tcc ctg atg agc tac aag tcc atc ctg gtg atg cgc 432 Arg Trp Gln Arg Ser Leu Met Ser Tyr Lys Ser Ile Leu Val Met Arg 130 135 140 tcc atc cct gag ctg ctg cgg ggc cca ccc gac agc ccc tcc ctc ggc 480 Ser Ile Pro Glu Leu Leu Arg Gly Pro Pro Asp Ser Pro Ser Leu Gly 145 150 155 160 gta gcc cgg cac ctc tgc agg gac act ggt ggc tcc tcc agt tcc tcc 528 Val Ala Arg His Leu Cys Arg Asp Thr Gly Gly Ser Ser Ser Ser Ser 165 170 175 tcc tcc tct gac aac gag ctc gcc cca ttt gcc cgg gct aag tca ctg 576 Ser Ser Ser Asp Asn Glu Leu Ala Pro Phe Ala Arg Ala Lys Ser Leu 180 185 190 cca ccc tcc ccg gtg aca cac tca cca ctg ctg cac ccc cgg ggc ttc 624 Pro Pro Ser Pro Val Thr His Ser Pro Leu Leu His Pro Arg Gly Phe 195 200 205 ctg cgg ccc tcg gcc agc ctg cct gag gaa gcc gag gcc agt gag cgc 672 Leu Arg Pro Ser Ala Ser Leu Pro Glu Glu Ala Glu Ala Ser Glu Arg 210 215 220 tcc acc gag gcc cca gct ccg cct gca tct ccc gag ggt gcc ggg cca 720 Ser Thr Glu Ala Pro Ala Pro Pro Ala Ser Pro Glu Gly Ala Gly Pro 225 230 235 240 ccg gcc gcc cag ggc tgc gtg ccc cgg cac agc gtc atc cgc agc ctg 768 Pro Ala Ala Gln Gly Cys Val Pro Arg His Ser Val Ile Arg Ser Leu 245 250 255 ttc tac cac cag gcg ggt gag agc cct gag cac ggg gcc ctg gcc ccg 816 Phe Tyr His Gln Ala Gly Glu Ser Pro Glu His Gly Ala Leu Ala Pro 260 265 270 ggg agc agg cgg cac ccg gcc cgg cgg cgg cac ctg ctg aag ggc ggg 864 Gly Ser Arg Arg His Pro Ala Arg Arg Arg His Leu Leu Lys Gly Gly 275 280 285 tac att gcg ggg gcg ctg cca ggc ctg cgc gag cca ctg atg gag cac 912 Tyr Ile Ala Gly Ala Leu Pro Gly Leu Arg Glu Pro Leu Met Glu His 290 295 300 cgc gtg ctg gag gag gag gcc gcc agg gag gag cag gcc acc ctc ctg 960 Arg Val Leu Glu Glu Glu Ala Ala Arg Glu Glu Gln Ala Thr Leu Leu 305 310 315 320 gcc aaa gcc ccc tca ttc gag act gcc ctc cgg ctg cct gcc tct ggc 1008 Ala Lys Ala Pro Ser Phe Glu Thr Ala Leu Arg Leu Pro Ala Ser Gly 325 330 335 acc cac ttg gcc cct ggc cac agc cac tcc ctg gaa cat gac tct ccg 1056 Thr His Leu Ala Pro Gly His Ser His Ser Leu Glu His Asp Ser Pro 340 345 350 agc acc ccc cgc ccc tcc tcg gag gcc tgc ggt gag gca cag cga ctg 1104 Ser Thr Pro Arg Pro Ser Ser Glu Ala Cys Gly Glu Ala Gln Arg Leu 355 360 365 cct tca gcc ccc tcc ggg ggg gcc cct atc agg gac atg ggg cac cct 1152 Pro Ser Ala Pro Ser Gly Gly Ala Pro Ile Arg Asp Met Gly His Pro 370 375 380 cag ggc tcc aag cag ctt cca tcc act ggt ggc cac cca ggc act gct 1200 Gln Gly Ser Lys Gln Leu Pro Ser Thr Gly Gly His Pro Gly Thr Ala 385 390 395 400 cag cca gag agg cca tcc ccg gac agc cct tgg ggg cag cca gcc cct 1248 Gln Pro Glu Arg Pro Ser Pro Asp Ser Pro Trp Gly Gln Pro Ala Pro 405 410 415 ttc tgc cac ccc aag cag ggt tct gcc ccc cag gag ggc tgc agc ccc 1296 Phe Cys His Pro Lys Gln Gly Ser Ala Pro Gln Glu Gly Cys Ser Pro 420 425 430 cac cca gca gtt gcc cca tgc cct cct ggc tcc ttc cct cca gga tct 1344 His Pro Ala Val Ala Pro Cys Pro Pro Gly Ser Phe Pro Pro Gly Ser 435 440 445 tgc aaa gag gcc ccc tta gta ccc tca agc ccc ttc ttg gga cag ccc 1392 Cys Lys Glu Ala Pro Leu Val Pro Ser Ser Pro Phe Leu Gly Gln Pro 450 455 460 cag gca ccc cct gcc cct gcc aaa gca agc ccc cca ttg gac tct aag 1440 Gln Ala Pro Pro Ala Pro Ala Lys Ala Ser Pro Pro Leu Asp Ser Lys 465 470 475 480 atg ggg cct gga gac atc tct ctt cct ggg agg cca aaa ccc ggc ccc 1488 Met Gly Pro Gly Asp Ile Ser Leu Pro Gly Arg Pro Lys Pro Gly Pro 485 490 495 tgc agt tcc cca ggg tca gcc tcc cag gcg agc tct tcc caa gtg agc 1536 Cys Ser Ser Pro Gly Ser Ala Ser Gln Ala Ser Ser Ser Gln Val Ser 500 505 510 tcc ctc agg gtg ggc tcc tcc cag gtg ggc aca gag cct ggc ccc tcc 1584 Ser Leu Arg Val Gly Ser Ser Gln Val Gly Thr Glu Pro Gly Pro Ser 515 520 525 ctg gat gcg gag ggc tgg acc cag gag gct gag gat ctg tcc gac tcc 1632 Leu Asp Ala Glu Gly Trp Thr Gln Glu Ala Glu Asp Leu Ser Asp Ser 530 535 540 aca ccc acc ttg cag cgg cct cag gaa cag gcg acc atg cgc aag ttc 1680 Thr Pro Thr Leu Gln Arg Pro Gln Glu Gln Ala Thr Met Arg Lys Phe 545 550 555 560 tcc ctg ggt ggt cgc ggg ggc tac gca ggc gtg gct ggc tat ggc acc 1728 Ser Leu Gly Gly Arg Gly Gly Tyr Ala Gly Val Ala Gly Tyr Gly Thr 565 570 575 ttt gcc ttt ggt gga gat gca ggg ggc atg ctg ggg cag ggg ccc atg 1776 Phe Ala Phe Gly Gly Asp Ala Gly Gly Met Leu Gly Gln Gly Pro Met 580 585 590 tgg gcc agg ata gcc tgg gct gtg tcc cag tca gag gag gag gag cag 1824 Trp Ala Arg Ile Ala Trp Ala Val Ser Gln Ser Glu Glu Glu Glu Gln 595 600 605 gag gag gcc agg gct gag tcc cag tcg gag gag cag cag gag gcc agg 1872 Glu Glu Ala Arg Ala Glu Ser Gln Ser Glu Glu Gln Gln Glu Ala Arg 610 615 620 gct gag agc cca ctg ccc cag gtc agt gca agg cct gtg cct gag gtc 1920 Ala Glu Ser Pro Leu Pro Gln Val Ser Ala Arg Pro Val Pro Glu Val 625 630 635 640 ggc agg gct ccc acc agg agc tct cca gag ccc acc cca tgg gag gac 1968 Gly Arg Ala Pro Thr Arg Ser Ser Pro Glu Pro Thr Pro Trp Glu Asp 645 650 655 atc ggg cag gtc tcc ctg gtg cag atc cgg gac ctg tca ggt gat gcg 2016 Ile Gly Gln Val Ser Leu Val Gln Ile Arg Asp Leu Ser Gly Asp Ala 660 665 670 gag gcg gcc gac aca ata tcc ctg gac att tcc gag gtg gac ccc gcc 2064 Glu Ala Ala Asp Thr Ile Ser Leu Asp Ile Ser Glu Val Asp Pro Ala 675 680 685 tac ctc aac ctc tca gac ctg tac gat atc aag tac ctc cca ttc gag 2112 Tyr Leu Asn Leu Ser Asp Leu Tyr Asp Ile Lys Tyr Leu Pro Phe Glu 690 695 700 ttt atg atc ttc agg aaa gtc ccc aag tcc gct cag cca gag ccg ccc 2160 Phe Met Ile Phe Arg Lys Val Pro Lys Ser Ala Gln Pro Glu Pro Pro 705 710 715 720 tcc ccc atg gct gag gag gag ctg gcc gag ttc ccg gag ccc acg tgg 2208 Ser Pro Met Ala Glu Glu Glu Leu Ala Glu Phe Pro Glu Pro Thr Trp 725 730 735 ccc tgg cca ggt gaa ctg ggc ccc cac gca ggc ctg gag atc aca gag 2256 Pro Trp Pro Gly Glu Leu Gly Pro His Ala Gly Leu Glu Ile Thr Glu 740 745 750 gag tca gag gat gtg gac gcg ctg ctg gca gag gct gcc gtg ggc agg 2304 Glu Ser Glu Asp Val Asp Ala Leu Leu Ala Glu Ala Ala Val Gly Arg 755 760 765 aag cgc aag tgg tcc tcg ccg tca cgc agc ctc ttc cac ttc cct ggg 2352 Lys Arg Lys Trp Ser Ser Pro Ser Arg Ser Leu Phe His Phe Pro Gly 770 775 780 agg cac ctg ccg ctg gat gag cct gca gag ctg ggg ctg cgt gag aga 2400 Arg His Leu Pro Leu Asp Glu Pro Ala Glu Leu Gly Leu Arg Glu Arg 785 790 795 800 gtg aag gcc tcc gtg gag cac atc tcc cgg atc ctg aag ggc agg ccg 2448 Val Lys Ala Ser Val Glu His Ile Ser Arg Ile Leu Lys Gly Arg Pro 805 810 815 gaa ggt ctg gag aag gag ggg ccc ccc agg aag aag cca ggc ctt gct 2496 Glu Gly Leu Glu Lys Glu Gly Pro Pro Arg Lys Lys Pro Gly Leu Ala 820 825 830 tcc ttc cgg ctc tca ggt ctg aag agc tgg gac cga gcg ccg aca ttc 2544 Ser Phe Arg Leu Ser Gly Leu Lys Ser Trp Asp Arg Ala Pro Thr Phe 835 840 845 cta agg gag ctc tca gat gag act gtg gtc ctg ggc cag tca gtg aca 2592 Leu Arg Glu Leu Ser Asp Glu Thr Val Val Leu Gly Gln Ser Val Thr 850 855 860 ctg gcc tgc cag gtg tca gcc cag cca gct gcc cag gcc acc tgg agc 2640 Leu Ala Cys Gln Val Ser Ala Gln Pro Ala Ala Gln Ala Thr Trp Ser 865 870 875 880 aaa gac gga gcc ccc ctg gag agc agc agc cgt gtc ctc atc tct gcc 2688 Lys Asp Gly Ala Pro Leu Glu Ser Ser Ser Arg Val Leu Ile Ser Ala 885 890 895 acc ctc aag aac ttc cag ctt ctg acc atc ctg gtg gtg gtg gct gag 2736 Thr Leu Lys Asn Phe Gln Leu Leu Thr Ile Leu Val Val Val Ala Glu 900 905 910 gac ctg ggt gtg tac acc tgc agc gtg agc aat gcg ctg ggg aca gtg 2784 Asp Leu Gly Val Tyr Thr Cys Ser Val Ser Asn Ala Leu Gly Thr Val 915 920 925 acc acc acg ggc gtc ctc cgg aag gca gag cgc ccc tca tct tcg cca 2832 Thr Thr Thr Gly Val Leu Arg Lys Ala Glu Arg Pro Ser Ser Ser Pro 930 935 940 tgc ccg gat atc ggg gag gtg tac gcg gat ggg gtg ctg ctg gtc tgg 2880 Cys Pro Asp Ile Gly Glu Val Tyr Ala Asp Gly Val Leu Leu Val Trp 945 950 955 960 aag ccc gtg gaa tcc tac ggc cct gtg acc tac att gtg cag tgc agc 2928 Lys Pro Val Glu Ser Tyr Gly Pro Val Thr Tyr Ile Val Gln Cys Ser 965 970 975 cta gaa ggc ggc agc tgg acc aca ctg gcc tcc gac atc ttt gac tgc 2976 Leu Glu Gly Gly Ser Trp Thr Thr Leu Ala Ser Asp Ile Phe Asp Cys 980 985 990 tgc tac ctg acc agc aag ctc tcc cgg ggt ggc acc tac acc ttc cgc 3024 Cys Tyr Leu Thr Ser Lys Leu Ser Arg Gly Gly Thr Tyr Thr Phe Arg 995 1000 1005 acg gca tgt gtc agc aag gca gga atg ggt ccc tac agc agc ccc tcg 3072 Thr Ala Cys Val Ser Lys Ala Gly Met Gly Pro Tyr Ser Ser Pro Ser 1010 1015 1020 gag caa gtc ctc ctg gga ggg ccc agc cac ctg gcc tct gag gag gag 3120 Glu Gln Val Leu Leu Gly Gly Pro Ser His Leu Ala Ser Glu Glu Glu 1025 1030 1035 1040 agc cag ggg cgg tca gcc caa ccc ctg ccc agc aca aag acc ttc gca 3168 Ser Gln Gly Arg Ser Ala Gln Pro Leu Pro Ser Thr Lys Thr Phe Ala 1045 1050 1055 ttc cag aca cag atc cag agg ggc cgc ttc agc gtg gtg cgg caa tgc 3216 Phe Gln Thr Gln Ile Gln Arg Gly Arg Phe Ser Val Val Arg Gln Cys 1060 1065 1070 tgg gag aag gcc agc ggg cgg gcg ctg gcc gcc aag atc atc ccc tac 3264 Trp Glu Lys Ala Ser Gly Arg Ala Leu Ala Ala Lys Ile Ile Pro Tyr 1075 1080 1085 cac ccc aag gac aag aca gca gtg ctg cgc gaa tac gag gcc ctc aag 3312 His Pro Lys Asp Lys Thr Ala Val Leu Arg Glu Tyr Glu Ala Leu Lys 1090 1095 1100 ggc ctg cgc cac ccg cac ctg gcc cag ctg cac gca gcc tac ctc agc 3360 Gly Leu Arg His Pro His Leu Ala Gln Leu His Ala Ala Tyr Leu Ser 1105 1110 1115 1120 ccc cgg cac ctg gtg ctc atc ttg gag ctg tgc tct ggg ccc gag ctg 3408 Pro Arg His Leu Val Leu Ile Leu Glu Leu Cys Ser Gly Pro Glu Leu 1125 1130 1135 ctc ccc tgc ctg gcc gag agg gcc tcc tac tca gaa tcc gag gtg aag 3456 Leu Pro Cys Leu Ala Glu Arg Ala Ser Tyr Ser Glu Ser Glu Val Lys 1140 1145 1150 gac tac ctg tgg cag atg ttg agt gcc acc cag tac ctg cac aac cag 3504 Asp Tyr Leu Trp Gln Met Leu Ser Ala Thr Gln Tyr Leu His Asn Gln 1155 1160 1165 cac atc ctg cac ctg gac ctg agg tcc gag aac atg atc atc acc gaa 3552 His Ile Leu His Leu Asp Leu Arg Ser Glu Asn Met Ile Ile Thr Glu 1170 1175 1180 tac aac ctg ctc aag gtc gtg gac ctg ggc aat gca cag agc ctc agc 3600 Tyr Asn Leu Leu Lys Val Val Asp Leu Gly Asn Ala Gln Ser Leu Ser 1185 1190 1195 1200 cag gag aag gtg ctg ccc tca gac aag ttc aag gac tac cta gag acc 3648 Gln Glu Lys Val Leu Pro Ser Asp Lys Phe Lys Asp Tyr Leu Glu Thr 1205 1210 1215 atg gct cca gag ctc ctg gag ggc cag ggg gct gtt cca cag aca gac 3696 Met Ala Pro Glu Leu Leu Glu Gly Gln Gly Ala Val Pro Gln Thr Asp 1220 1225 1230 atc tgg gcc atc ggt gtg aca gcc ttc atc atg ctg agc gcc gag tac 3744 Ile Trp Ala Ile Gly Val Thr Ala Phe Ile Met Leu Ser Ala Glu Tyr 1235 1240 1245 ccg gtg agc agc gag ggt gca cgc gac ctg cag aga gga ctg cgc aag 3792 Pro Val Ser Ser Glu Gly Ala Arg Asp Leu Gln Arg Gly Leu Arg Lys 1250 1255 1260 ggg ctg gtc cgg ctg agc cgc tgc tac gcg ggg ctg tcc ggg ggc gcc 3840 Gly Leu Val Arg Leu Ser Arg Cys Tyr Ala Gly Leu Ser Gly Gly Ala 1265 1270 1275 1280 gtg gcc ttc ctg cgc agc act ctg tgc gcc cag ccc tgg ggc cgg ccc 3888 Val Ala Phe Leu Arg Ser Thr Leu Cys Ala Gln Pro Trp Gly Arg Pro 1285 1290 1295 tgc gcg tcc agc tgc ctg cag tgc ccg tgg cta aca gag gag ggc ccg 3936 Cys Ala Ser Ser Cys Leu Gln Cys Pro Trp Leu Thr Glu Glu Gly Pro 1300 1305 1310 gcc tgt tcg cgg ccc gcg ccc gtg acc ttc cct acc gcg cgg ctg cgc 3984 Ala Cys Ser Arg Pro Ala Pro Val Thr Phe Pro Thr Ala Arg Leu Arg 1315 1320 1325 gtc ttc gtg cgc aat cgc gag aag aga cgc gcg ctg ctg tac aag agg 4032 Val Phe Val Arg Asn Arg Glu Lys Arg Arg Ala Leu Leu Tyr Lys Arg 1330 1335 1340 cac aac ctg gcc cag gtg cgc tgagggtcgc cccggccaca cccttggtct 4083 His Asn Leu Ala Gln Val Arg 1345 1350 ccccgctggg ggtcgctgca gacgcgccaa taaaaacgca cagccgggcg agaaaaaaaa 4143 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 4175 3 5007 DNA Homo sapiens CDS (97)...(4926) 3 ctgctctggg acgtccacag ccacgtggtc agagagacca cacagaggac ctacacatac 60 caggccatcg acacgcacac cgcacggccc ccatcc atg cag gta acc atc gag 114 Met Gln Val Thr Ile Glu 1 5 gat gtg cag gca cag aca ggc gga acg gcc caa ttc gag gct atc att 162 Asp Val Gln Ala Gln Thr Gly Gly Thr Ala Gln Phe Glu Ala Ile Ile 10 15 20 gag ggc gac cca cag ccc tcg gtg acc tgg tac aag gac agc gtc cag 210 Glu Gly Asp Pro Gln Pro Ser Val Thr Trp Tyr Lys Asp Ser Val Gln 25 30 35 ctg gtg gac agc acc cgg ctt agc cag cag caa gaa ggc acc aca tac 258 Leu Val Asp Ser Thr Arg Leu Ser Gln Gln Gln Glu Gly Thr Thr Tyr 40 45 50 tcc ctg gtg ctg agg cat gtg gcc tcg aag gat gcc ggc gtt tac acc 306 Ser Leu Val Leu Arg His Val Ala Ser Lys Asp Ala Gly Val Tyr Thr 55 60 65 70 tgc ctg gcc caa aac act ggt ggc cag gtg ctc tgc aag gca gag ctg 354 Cys Leu Ala Gln Asn Thr Gly Gly Gln Val Leu Cys Lys Ala Glu Leu 75 80 85 ctg gtg ctt ggg ggg gac aat gag ccg gac tca gag aag caa agc cac 402 Leu Val Leu Gly Gly Asp Asn Glu Pro Asp Ser Glu Lys Gln Ser His 90 95 100 cgg agg aag ctg cac tcc ttc tat gag gtc aag gag gag att gga agg 450 Arg Arg Lys Leu His Ser Phe Tyr Glu Val Lys Glu Glu Ile Gly Arg 105 110 115 ggc gtg ttt ggc ttc gta aaa aga gtg cag cac aaa gga aac aag atc 498 Gly Val Phe Gly Phe Val Lys Arg Val Gln His Lys Gly Asn Lys Ile 120 125 130 ttg tgc gct gcc aag ttc atc ccc cta cgg agc aga act cgg gcc cag 546 Leu Cys Ala Ala Lys Phe Ile Pro Leu Arg Ser Arg Thr Arg Ala Gln 135 140 145 150 gca tac agg gag cga gac atc ctg gcc gcg ctg agc cac ccg ctg gtc 594 Ala Tyr Arg Glu Arg Asp Ile Leu Ala Ala Leu Ser His Pro Leu Val 155 160 165 acg ggg ctg ctg gac cag ttt gag acc cgc aag acc ctc atc ctc atc 642 Thr Gly Leu Leu Asp Gln Phe Glu Thr Arg Lys Thr Leu Ile Leu Ile 170 175 180 ctg gag ctg tgc tca tcc gag gag ctg ctg gac cgc ctg tac agg aag 690 Leu Glu Leu Cys Ser Ser Glu Glu Leu Leu Asp Arg Leu Tyr Arg Lys 185 190 195 ggc gtg gtg acg gag gcc gag gtc aag gtc tac atc cag cag ctg gtg 738 Gly Val Val Thr Glu Ala Glu Val Lys Val Tyr Ile Gln Gln Leu Val 200 205 210 gag ggg ctg cac tac ctg cac agc cat ggc gtt ctc cac ctg gac ata 786 Glu Gly Leu His Tyr Leu His Ser His Gly Val Leu His Leu Asp Ile 215 220 225 230 aag ccc tct aac atc ctg atg gtg cat cct gcc cgg gaa gac att aaa 834 Lys Pro Ser Asn Ile Leu Met Val His Pro Ala Arg Glu Asp Ile Lys 235 240 245 atc tgc gac ttt ggc ttt gcc cag aac atc acc cca gca gag ctg cag 882 Ile Cys Asp Phe Gly Phe Ala Gln Asn Ile Thr Pro Ala Glu Leu Gln 250 255 260 ttc agc cag tac ggc tcc cct gag ttc gtc tcc ccc gag atc atc cag 930 Phe Ser Gln Tyr Gly Ser Pro Glu Phe Val Ser Pro Glu Ile Ile Gln 265 270 275 cag aac cct gtg agc gaa gcc tcc gac att tgg gcc atg ggt gtc atc 978 Gln Asn Pro Val Ser Glu Ala Ser Asp Ile Trp Ala Met Gly Val Ile 280 285 290 tcc tac ctc agc ctg acc tgc tca tcc cca ttt gcc ggc gag agt gac 1026 Ser Tyr Leu Ser Leu Thr Cys Ser Ser Pro Phe Ala Gly Glu Ser Asp 295 300 305 310 cgt gcc acc ctc ctg aac gtc ctg gag ggg cgc gtg tca tgg agc agc 1074 Arg Ala Thr Leu Leu Asn Val Leu Glu Gly Arg Val Ser Trp Ser Ser 315 320 325 ccc atg gct gcc cac ctc agc gaa gac gcc aaa gac ttc atc aag gct 1122 Pro Met Ala Ala His Leu Ser Glu Asp Ala Lys Asp Phe Ile Lys Ala 330 335 340 acg ctg cag aga gcc cct cag gcc cgg cct agt gcg gcc cag tgc ctc 1170 Thr Leu Gln Arg Ala Pro Gln Ala Arg Pro Ser Ala Ala Gln Cys Leu 345 350 355 tcc cac ccc tgg ttc ctg aaa tcc atg cct gcg gag gag gcc cac ttc 1218 Ser His Pro Trp Phe Leu Lys Ser Met Pro Ala Glu Glu Ala His Phe 360 365 370 atc aac acc aag cag ctc aag ttc ctc ctg gcc cga agt cgc tgg cag 1266 Ile Asn Thr Lys Gln Leu Lys Phe Leu Leu Ala Arg Ser Arg Trp Gln 375 380 385 390 cgt tcc ctg atg agc tac aag tcc atc ctg gtg atg cgc tcc atc cct 1314 Arg Ser Leu Met Ser Tyr Lys Ser Ile Leu Val Met Arg Ser Ile Pro 395 400 405 gag ctg ctg cgg ggc cca ccc gac agc ccc tcc ctc ggc gta gcc cgg 1362 Glu Leu Leu Arg Gly Pro Pro Asp Ser Pro Ser Leu Gly Val Ala Arg 410 415 420 cac ctc tgc agg gac act ggt ggc tcc tcc agt tcc tcc tcc tcc tct 1410 His Leu Cys Arg Asp Thr Gly Gly Ser Ser Ser Ser Ser Ser Ser Ser 425 430 435 gac aac gag ctc gcc cca ttt gcc cgg gct aag tca ctg cca ccc tcc 1458 Asp Asn Glu Leu Ala Pro Phe Ala Arg Ala Lys Ser Leu Pro Pro Ser 440 445 450 ccg gtg aca cac tca cca ctg ctg cac ccc cgg ggc ttc ctg cgg ccc 1506 Pro Val Thr His Ser Pro Leu Leu His Pro Arg Gly Phe Leu Arg Pro 455 460 465 470 tcg gcc agc ctg cct gag gaa gcc gag gcc agt gag cgc tcc acc gag 1554 Ser Ala Ser Leu Pro Glu Glu Ala Glu Ala Ser Glu Arg Ser Thr Glu 475 480 485 gcc cca gct ccg cct gca tct ccc gag ggt gcc ggg cca ccg gcc gcc 1602 Ala Pro Ala Pro Pro Ala Ser Pro Glu Gly Ala Gly Pro Pro Ala Ala 490 495 500 cag ggc tgc gtg ccc cgg cac agc gtc atc cgc agc ctg ttc tac cac 1650 Gln Gly Cys Val Pro Arg His Ser Val Ile Arg Ser Leu Phe Tyr His 505 510 515 cag gcg ggt gag agc cct gag cac ggg gcc ctg gcc ccg ggg agc agg 1698 Gln Ala Gly Glu Ser Pro Glu His Gly Ala Leu Ala Pro Gly Ser Arg 520 525 530 cgg cac ccg gcc cgg cgg cgg cac ctg ctg aag ggc ggg tac att gcg 1746 Arg His Pro Ala Arg Arg Arg His Leu Leu Lys Gly Gly Tyr Ile Ala 535 540 545 550 ggg gcg ctg cca ggc ctg cgc gag cca ctg atg gag cac cgc gtg ctg 1794 Gly Ala Leu Pro Gly Leu Arg Glu Pro Leu Met Glu His Arg Val Leu 555 560 565 gag gag gag gcc gcc agg gag gag cag gcc acc ctc ctg gcc aaa gcc 1842 Glu Glu Glu Ala Ala Arg Glu Glu Gln Ala Thr Leu Leu Ala Lys Ala 570 575 580 ccc tca ttc gag act gcc ctc cgg ctg cct gcc tct ggc acc cac ttg 1890 Pro Ser Phe Glu Thr Ala Leu Arg Leu Pro Ala Ser Gly Thr His Leu 585 590 595 gcc cct ggc cac agc cac tcc ctg gaa cat gac tct ccg agc acc ccc 1938 Ala Pro Gly His Ser His Ser Leu Glu His Asp Ser Pro Ser Thr Pro 600 605 610 cgc ccc tcc tcg gag gcc tgc ggt gag gca cag cga ctg cct tca gcc 1986 Arg Pro Ser Ser Glu Ala Cys Gly Glu Ala Gln Arg Leu Pro Ser Ala 615 620 625 630 ccc tcc ggg ggg gcc cct atc agg gac atg ggg cac cct cag ggc tcc 2034 Pro Ser Gly Gly Ala Pro Ile Arg Asp Met Gly His Pro Gln Gly Ser 635 640 645 aag cag ctt cca tcc act ggt ggc cac cca ggc act gct cag cca gag 2082 Lys Gln Leu Pro Ser Thr Gly Gly His Pro Gly Thr Ala Gln Pro Glu 650 655 660 agg cca tcc ccg gac agc cct tgg ggg cag cca gcc cct ttc tgc cac 2130 Arg Pro Ser Pro Asp Ser Pro Trp Gly Gln Pro Ala Pro Phe Cys His 665 670 675 ccc aag cag ggt tct gcc ccc cag gag ggc tgc agc ccc cac cca gca 2178 Pro Lys Gln Gly Ser Ala Pro Gln Glu Gly Cys Ser Pro His Pro Ala 680 685 690 gtt gcc cca tgc cct cct ggc tcc ttc cct cca gga tct tgc aaa gag 2226 Val Ala Pro Cys Pro Pro Gly Ser Phe Pro Pro Gly Ser Cys Lys Glu 695 700 705 710 gcc ccc tta gta ccc tca agc ccc ttc ttg gga cag ccc cag gca ccc 2274 Ala Pro Leu Val Pro Ser Ser Pro Phe Leu Gly Gln Pro Gln Ala Pro 715 720 725 cct gcc cct gcc aaa gca agc ccc cca ttg gac tct aag atg ggg cct 2322 Pro Ala Pro Ala Lys Ala Ser Pro Pro Leu Asp Ser Lys Met Gly Pro 730 735 740 gga gac atc tct ctt cct ggg agg cca aaa ccc ggc ccc tgc agt tcc 2370 Gly Asp Ile Ser Leu Pro Gly Arg Pro Lys Pro Gly Pro Cys Ser Ser 745 750 755 cca ggg tca gcc tcc cag gcg agc tct tcc caa gtg agc tcc ctc agg 2418 Pro Gly Ser Ala Ser Gln Ala Ser Ser Ser Gln Val Ser Ser Leu Arg 760 765 770 gtg ggc tcc tcc cag gtg ggc aca gag cct ggc ccc tcc ctg gat gcg 2466 Val Gly Ser Ser Gln Val Gly Thr Glu Pro Gly Pro Ser Leu Asp Ala 775 780 785 790 gag ggc tgg acc cag gag gct gag gat ctg tcc gac tcc aca ccc acc 2514 Glu Gly Trp Thr Gln Glu Ala Glu Asp Leu Ser Asp Ser Thr Pro Thr 795 800 805 ttg cag cgg cct cag gaa cag gcg acc atg cgc aag ttc tcc ctg ggt 2562 Leu Gln Arg Pro Gln Glu Gln Ala Thr Met Arg Lys Phe Ser Leu Gly 810 815 820 ggt cgc ggg ggc tac gca ggc gtg gct ggc tat ggc acc ttt gcc ttt 2610 Gly Arg Gly Gly Tyr Ala Gly Val Ala Gly Tyr Gly Thr Phe Ala Phe 825 830 835 ggt gga gat gca ggg ggc atg ctg ggg cag ggg ccc atg tgg gcc agg 2658 Gly Gly Asp Ala Gly Gly Met Leu Gly Gln Gly Pro Met Trp Ala Arg 840 845 850 ata gcc tgg gct gtg tcc cag tca gag gag gag gag cag gag gag gcc 2706 Ile Ala Trp Ala Val Ser Gln Ser Glu Glu Glu Glu Gln Glu Glu Ala 855 860 865 870 agg gct gag tcc cag tcg gag gag cag cag gag gcc agg gct gag agc 2754 Arg Ala Glu Ser Gln Ser Glu Glu Gln Gln Glu Ala Arg Ala Glu Ser 875 880 885 cca ctg ccc cag gtc agt gca agg cct gtg cct gag gtc ggc agg gct 2802 Pro Leu Pro Gln Val Ser Ala Arg Pro Val Pro Glu Val Gly Arg Ala 890 895 900 ccc acc agg agc tct cca gag ccc acc cca tgg gag gac atc ggg cag 2850 Pro Thr Arg Ser Ser Pro Glu Pro Thr Pro Trp Glu Asp Ile Gly Gln 905 910 915 gtc tcc ctg gtg cag atc cgg gac ctg tca ggt gat gcg gag gcg gcc 2898 Val Ser Leu Val Gln Ile Arg Asp Leu Ser Gly Asp Ala Glu Ala Ala 920 925 930 gac aca ata tcc ctg gac att tcc gag gtg gac ccc gcc tac ctc aac 2946 Asp Thr Ile Ser Leu Asp Ile Ser Glu Val Asp Pro Ala Tyr Leu Asn 935 940 945 950 ctc tca gac ctg tac gat atc aag tac ctc cca ttc gag ttt atg atc 2994 Leu Ser Asp Leu Tyr Asp Ile Lys Tyr Leu Pro Phe Glu Phe Met Ile 955 960 965 ttc agg aaa gtc ccc aag tcc gct cag cca gag ccg ccc tcc ccc atg 3042 Phe Arg Lys Val Pro Lys Ser Ala Gln Pro Glu Pro Pro Ser Pro Met 970 975 980 gct gag gag gag ctg gcc gag ttc ccg gag ccc acg tgg ccc tgg cca 3090 Ala Glu Glu Glu Leu Ala Glu Phe Pro Glu Pro Thr Trp Pro Trp Pro 985 990 995 ggt gaa ctg ggc ccc cac gca ggc ctg gag atc aca gag gag tca gag 3138 Gly Glu Leu Gly Pro His Ala Gly Leu Glu Ile Thr Glu Glu Ser Glu 1000 1005 1010 gat gtg gac gcg ctg ctg gca gag gct gcc gtg ggc agg aag cgc aag 3186 Asp Val Asp Ala Leu Leu Ala Glu Ala Ala Val Gly Arg Lys Arg Lys 1015 1020 1025 1030 tgg tcc tcg ccg tca cgc agc ctc ttc cac ttc cct ggg agg cac ctg 3234 Trp Ser Ser Pro Ser Arg Ser Leu Phe His Phe Pro Gly Arg His Leu 1035 1040 1045 ccg ctg gat gag cct gca gag ctg ggg ctg cgt gag aga gtg aag gcc 3282 Pro Leu Asp Glu Pro Ala Glu Leu Gly Leu Arg Glu Arg Val Lys Ala 1050 1055 1060 tcc gtg gag cac atc tcc cgg atc ctg aag ggc agg ccg gaa ggt ctg 3330 Ser Val Glu His Ile Ser Arg Ile Leu Lys Gly Arg Pro Glu Gly Leu 1065 1070 1075 gag aag gag ggg ccc ccc agg aag aag cca ggc ctt gct tcc ttc cgg 3378 Glu Lys Glu Gly Pro Pro Arg Lys Lys Pro Gly Leu Ala Ser Phe Arg 1080 1085 1090 ctc tca ggt ctg aag agc tgg gac cga gcg ccg aca ttc cta agg gag 3426 Leu Ser Gly Leu Lys Ser Trp Asp Arg Ala Pro Thr Phe Leu Arg Glu 1095 1100 1105 1110 ctc tca gat gag act gtg gtc ctg ggc cag tca gtg aca ctg gcc tgc 3474 Leu Ser Asp Glu Thr Val Val Leu Gly Gln Ser Val Thr Leu Ala Cys 1115 1120 1125 cag gtg tca gcc cag cca gct gcc cag gcc acc tgg agc aaa gac gga 3522 Gln Val Ser Ala Gln Pro Ala Ala Gln Ala Thr Trp Ser Lys Asp Gly 1130 1135 1140 gcc ccc ctg gag agc agc agc cgt gtc ctc atc tct gcc acc ctc aag 3570 Ala Pro Leu Glu Ser Ser Ser Arg Val Leu Ile Ser Ala Thr Leu Lys 1145 1150 1155 aac ttc cag ctt ctg acc atc ctg gtg gtg gtg gct gag gac ctg ggt 3618 Asn Phe Gln Leu Leu Thr Ile Leu Val Val Val Ala Glu Asp Leu Gly 1160 1165 1170 gtg tac acc tgc agc gtg agc aat gcg ctg ggg aca gtg acc acc acg 3666 Val Tyr Thr Cys Ser Val Ser Asn Ala Leu Gly Thr Val Thr Thr Thr 1175 1180 1185 1190 ggc gtc ctc cgg aag gca gag cgc ccc tca tct tcg cca tgc ccg gat 3714 Gly Val Leu Arg Lys Ala Glu Arg Pro Ser Ser Ser Pro Cys Pro Asp 1195 1200 1205 atc ggg gag gtg tac gcg gat ggg gtg ctg ctg gtc tgg aag ccc gtg 3762 Ile Gly Glu Val Tyr Ala Asp Gly Val Leu Leu Val Trp Lys Pro Val 1210 1215 1220 gaa tcc tac ggc cct gtg acc tac att gtg cag tgc agc cta gaa ggc 3810 Glu Ser Tyr Gly Pro Val Thr Tyr Ile Val Gln Cys Ser Leu Glu Gly 1225 1230 1235 ggc agc tgg acc aca ctg gcc tcc gac atc ttt gac tgc tgc tac ctg 3858 Gly Ser Trp Thr Thr Leu Ala Ser Asp Ile Phe Asp Cys Cys Tyr Leu 1240 1245 1250 acc agc aag ctc tcc cgg ggt ggc acc tac acc ttc cgc acg gca tgt 3906 Thr Ser Lys Leu Ser Arg Gly Gly Thr Tyr Thr Phe Arg Thr Ala Cys 1255 1260 1265 1270 gtc agc aag gca gga atg ggt ccc tac agc agc ccc tcg gag caa gtc 3954 Val Ser Lys Ala Gly Met Gly Pro Tyr Ser Ser Pro Ser Glu Gln Val 1275 1280 1285 ctc ctg gga gcg ccc agc cac ctg gcc tct gag gag gag agc cag ggg 4002 Leu Leu Gly Ala Pro Ser His Leu Ala Ser Glu Glu Glu Ser Gln Gly 1290 1295 1300 cgg tca gcc caa ccc ctg ccc agc aca aag acc ttc gca ttc cag aca 4050 Arg Ser Ala Gln Pro Leu Pro Ser Thr Lys Thr Phe Ala Phe Gln Thr 1305 1310 1315 cag atc cag agg ggc cgc ttc agc gtg gtg cgg caa tgc tgg gag aag 4098 Gln Ile Gln Arg Gly Arg Phe Ser Val Val Arg Gln Cys Trp Glu Lys 1320 1325 1330 gcc agc ggg cgg gcg ctg gcc gcc aag atc atc ccc tac cac ccc aag 4146 Ala Ser Gly Arg Ala Leu Ala Ala Lys Ile Ile Pro Tyr His Pro Lys 1335 1340 1345 1350 gac aag aca gca gtg ctg cgc gaa tac gag gcc ctc aag ggc ctg cgc 4194 Asp Lys Thr Ala Val Leu Arg Glu Tyr Glu Ala Leu Lys Gly Leu Arg 1355 1360 1365 cac ccg cac ctg gcc cag ctg cac gca gcc tac ctc agc ccc cgg cac 4242 His Pro His Leu Ala Gln Leu His Ala Ala Tyr Leu Ser Pro Arg His 1370 1375 1380 ctg gtg ctc atc ttg gag ctg tgc tct ggg ccc gag ctg ctc ccc tgc 4290 Leu Val Leu Ile Leu Glu Leu Cys Ser Gly Pro Glu Leu Leu Pro Cys 1385 1390 1395 ctg gcc gag agg gcc tcc tac tca gaa tcc gag gtg aag gac tac ctg 4338 Leu Ala Glu Arg Ala Ser Tyr Ser Glu Ser Glu Val Lys Asp Tyr Leu 1400 1405 1410 tgg cag atg ttg agt gcc acc cag tac ctg cac aac cag cac atc ctg 4386 Trp Gln Met Leu Ser Ala Thr Gln Tyr Leu His Asn Gln His Ile Leu 1415 1420 1425 1430 cac ctg gac ctg agg tcc gag aac atg atc atc acc gaa tac aac ctg 4434 His Leu Asp Leu Arg Ser Glu Asn Met Ile Ile Thr Glu Tyr Asn Leu 1435 1440 1445 ctc aag gtc gtg gac ctg ggc aat gca cag agc ctc agc cag gag aag 4482 Leu Lys Val Val Asp Leu Gly Asn Ala Gln Ser Leu Ser Gln Glu Lys 1450 1455 1460 gtg ctg ccc tca gac aag ttc aag gac tac cta gag acc atg gct cca 4530 Val Leu Pro Ser Asp Lys Phe Lys Asp Tyr Leu Glu Thr Met Ala Pro 1465 1470 1475 gag ctc ctg gag ggc cag ggg gct gtt cca cag aca gac atc tgg gcc 4578 Glu Leu Leu Glu Gly Gln Gly Ala Val Pro Gln Thr Asp Ile Trp Ala 1480 1485 1490 atc ggt gtg aca gcc ttc atc atg ctg agc gcc gag tac ccg gtg agc 4626 Ile Gly Val Thr Ala Phe Ile Met Leu Ser Ala Glu Tyr Pro Val Ser 1495 1500 1505 1510 agc gag ggt gca cgc gac ctg cag aga gga ctg cgc aag ggg ctg gtc 4674 Ser Glu Gly Ala Arg Asp Leu Gln Arg Gly Leu Arg Lys Gly Leu Val 1515 1520 1525 cgg ctg agc cgc tgc tac gcg ggg ctg tcc ggg ggc gcc gtg gcc ttc 4722 Arg Leu Ser Arg Cys Tyr Ala Gly Leu Ser Gly Gly Ala Val Ala Phe 1530 1535 1540 ctg cgc agc act ctg tgc gcc cag ccc tgg ggc cgg ccc tgc gcg tcc 4770 Leu Arg Ser Thr Leu Cys Ala Gln Pro Trp Gly Arg Pro Cys Ala Ser 1545 1550 1555 agc tgc ctg cag tgc ccg tgg cta aca gag gag ggc ccg gcc tgt tcg 4818 Ser Cys Leu Gln Cys Pro Trp Leu Thr Glu Glu Gly Pro Ala Cys Ser 1560 1565 1570 cgg ccc gcg ccc gtg acc ttc cct acc gcg cgg ctg cgc gtc ttc gtg 4866 Arg Pro Ala Pro Val Thr Phe Pro Thr Ala Arg Leu Arg Val Phe Val 1575 1580 1585 1590 cgc aat cgc gag aag aga cgc gcg ctg ctg tac aag agg cac aac ctg 4914 Arg Asn Arg Glu Lys Arg Arg Ala Leu Leu Tyr Lys Arg His Asn Leu 1595 1600 1605 gcc cag gtg cgc tgagggtcgc cccggccaca cccttggtct ccccgctggg 4966 Ala Gln Val Arg 1610 ggtcgctgca gacgcgccaa taaaaacgca cagccgggcg a 5007 4 1610 PRT Homo sapiens 4 Met Gln Val Thr Ile Glu Asp Val Gln Ala Gln Thr Gly Gly Thr Ala 1 5 10 15 Gln Phe Glu Ala Ile Ile Glu Gly Asp Pro Gln Pro Ser Val Thr Trp 20 25 30 Tyr Lys Asp Ser Val Gln Leu Val Asp Ser Thr Arg Leu Ser Gln Gln 35 40 45 Gln Glu Gly Thr Thr Tyr Ser Leu Val Leu Arg His Val Ala Ser Lys 50 55 60 Asp Ala Gly Val Tyr Thr Cys Leu Ala Gln Asn Thr Gly Gly Gln Val 65 70 75 80 Leu Cys Lys Ala Glu Leu Leu Val Leu Gly Gly Asp Asn Glu Pro Asp 85 90 95 Ser Glu Lys Gln Ser His Arg Arg Lys Leu His Ser Phe Tyr Glu Val 100 105 110 Lys Glu Glu Ile Gly Arg Gly Val Phe Gly Phe Val Lys Arg Val Gln 115 120 125 His Lys Gly Asn Lys Ile Leu Cys Ala Ala Lys Phe Ile Pro Leu Arg 130 135 140 Ser Arg Thr Arg Ala Gln Ala Tyr Arg Glu Arg Asp Ile Leu Ala Ala 145 150 155 160 Leu Ser His Pro Leu Val Thr Gly Leu Leu Asp Gln Phe Glu Thr Arg 165 170 175 Lys Thr Leu Ile Leu Ile Leu Glu Leu Cys Ser Ser Glu Glu Leu Leu 180 185 190 Asp Arg Leu Tyr Arg Lys Gly Val Val Thr Glu Ala Glu Val Lys Val 195 200 205 Tyr Ile Gln Gln Leu Val Glu Gly Leu His Tyr Leu His Ser His Gly 210 215 220 Val Leu His Leu Asp Ile Lys Pro Ser Asn Ile Leu Met Val His Pro 225 230 235 240 Ala Arg Glu Asp Ile Lys Ile Cys Asp Phe Gly Phe Ala Gln Asn Ile 245 250 255 Thr Pro Ala Glu Leu Gln Phe Ser Gln Tyr Gly Ser Pro Glu Phe Val 260 265 270 Ser Pro Glu Ile Ile Gln Gln Asn Pro Val Ser Glu Ala Ser Asp Ile 275 280 285 Trp Ala Met Gly Val Ile Ser Tyr Leu Ser Leu Thr Cys Ser Ser Pro 290 295 300 Phe Ala Gly Glu Ser Asp Arg Ala Thr Leu Leu Asn Val Leu Glu Gly 305 310 315 320 Arg Val Ser Trp Ser Ser Pro Met Ala Ala His Leu Ser Glu Asp Ala 325 330 335 Lys Asp Phe Ile Lys Ala Thr Leu Gln Arg Ala Pro Gln Ala Arg Pro 340 345 350 Ser Ala Ala Gln Cys Leu Ser His Pro Trp Phe Leu Lys Ser Met Pro 355 360 365 Ala Glu Glu Ala His Phe Ile Asn Thr Lys Gln Leu Lys Phe Leu Leu 370 375 380 Ala Arg Ser Arg Trp Gln Arg Ser Leu Met Ser Tyr Lys Ser Ile Leu 385 390 395 400 Val Met Arg Ser Ile Pro Glu Leu Leu Arg Gly Pro Pro Asp Ser Pro 405 410 415 Ser Leu Gly Val Ala Arg His Leu Cys Arg Asp Thr Gly Gly Ser Ser 420 425 430 Ser Ser Ser Ser Ser Ser Asp Asn Glu Leu Ala Pro Phe Ala Arg Ala 435 440 445 Lys Ser Leu Pro Pro Ser Pro Val Thr His Ser Pro Leu Leu His Pro 450 455 460 Arg Gly Phe Leu Arg Pro Ser Ala Ser Leu Pro Glu Glu Ala Glu Ala 465 470 475 480 Ser Glu Arg Ser Thr Glu Ala Pro Ala Pro Pro Ala Ser Pro Glu Gly 485 490 495 Ala Gly Pro Pro Ala Ala Gln Gly Cys Val Pro Arg His Ser Val Ile 500 505 510 Arg Ser Leu Phe Tyr His Gln Ala Gly Glu Ser Pro Glu His Gly Ala 515 520 525 Leu Ala Pro Gly Ser Arg Arg His Pro Ala Arg Arg Arg His Leu Leu 530 535 540 Lys Gly Gly Tyr Ile Ala Gly Ala Leu Pro Gly Leu Arg Glu Pro Leu 545 550 555 560 Met Glu His Arg Val Leu Glu Glu Glu Ala Ala Arg Glu Glu Gln Ala 565 570 575 Thr Leu Leu Ala Lys Ala Pro Ser Phe Glu Thr Ala Leu Arg Leu Pro 580 585 590 Ala Ser Gly Thr His Leu Ala Pro Gly His Ser His Ser Leu Glu His 595 600 605 Asp Ser Pro Ser Thr Pro Arg Pro Ser Ser Glu Ala Cys Gly Glu Ala 610 615 620 Gln Arg Leu Pro Ser Ala Pro Ser Gly Gly Ala Pro Ile Arg Asp Met 625 630 635 640 Gly His Pro Gln Gly Ser Lys Gln Leu Pro Ser Thr Gly Gly His Pro 645 650 655 Gly Thr Ala Gln Pro Glu Arg Pro Ser Pro Asp Ser Pro Trp Gly Gln 660 665 670 Pro Ala Pro Phe Cys His Pro Lys Gln Gly Ser Ala Pro Gln Glu Gly 675 680 685 Cys Ser Pro His Pro Ala Val Ala Pro Cys Pro Pro Gly Ser Phe Pro 690 695 700 Pro Gly Ser Cys Lys Glu Ala Pro Leu Val Pro Ser Ser Pro Phe Leu 705 710 715 720 Gly Gln Pro Gln Ala Pro Pro Ala Pro Ala Lys Ala Ser Pro Pro Leu 725 730 735 Asp Ser Lys Met Gly Pro Gly Asp Ile Ser Leu Pro Gly Arg Pro Lys 740 745 750 Pro Gly Pro Cys Ser Ser Pro Gly Ser Ala Ser Gln Ala Ser Ser Ser 755 760 765 Gln Val Ser Ser Leu Arg Val Gly Ser Ser Gln Val Gly Thr Glu Pro 770 775 780 Gly Pro Ser Leu Asp Ala Glu Gly Trp Thr Gln Glu Ala Glu Asp Leu 785 790 795 800 Ser Asp Ser Thr Pro Thr Leu Gln Arg Pro Gln Glu Gln Ala Thr Met 805 810 815 Arg Lys Phe Ser Leu Gly Gly Arg Gly Gly Tyr Ala Gly Val Ala Gly 820 825 830 Tyr Gly Thr Phe Ala Phe Gly Gly Asp Ala Gly Gly Met Leu Gly Gln 835 840 845 Gly Pro Met Trp Ala Arg Ile Ala Trp Ala Val Ser Gln Ser Glu Glu 850 855 860 Glu Glu Gln Glu Glu Ala Arg Ala Glu Ser Gln Ser Glu Glu Gln Gln 865 870 875 880 Glu Ala Arg Ala Glu Ser Pro Leu Pro Gln Val Ser Ala Arg Pro Val 885 890 895 Pro Glu Val Gly Arg Ala Pro Thr Arg Ser Ser Pro Glu Pro Thr Pro 900 905 910 Trp Glu Asp Ile Gly Gln Val Ser Leu Val Gln Ile Arg Asp Leu Ser 915 920 925 Gly Asp Ala Glu Ala Ala Asp Thr Ile Ser Leu Asp Ile Ser Glu Val 930 935 940 Asp Pro Ala Tyr Leu Asn Leu Ser Asp Leu Tyr Asp Ile Lys Tyr Leu 945 950 955 960 Pro Phe Glu Phe Met Ile Phe Arg Lys Val Pro Lys Ser Ala Gln Pro 965 970 975 Glu Pro Pro Ser Pro Met Ala Glu Glu Glu Leu Ala Glu Phe Pro Glu 980 985 990 Pro Thr Trp Pro Trp Pro Gly Glu Leu Gly Pro His Ala Gly Leu Glu 995 1000 1005 Ile Thr Glu Glu Ser Glu Asp Val Asp Ala Leu Leu Ala Glu Ala Ala 1010 1015 1020 Val Gly Arg Lys Arg Lys Trp Ser Ser Pro Ser Arg Ser Leu Phe His 1025 1030 1035 1040 Phe Pro Gly Arg His Leu Pro Leu Asp Glu Pro Ala Glu Leu Gly Leu 1045 1050 1055 Arg Glu Arg Val Lys Ala Ser Val Glu His Ile Ser Arg Ile Leu Lys 1060 1065 1070 Gly Arg Pro Glu Gly Leu Glu Lys Glu Gly Pro Pro Arg Lys Lys Pro 1075 1080 1085 Gly Leu Ala Ser Phe Arg Leu Ser Gly Leu Lys Ser Trp Asp Arg Ala 1090 1095 1100 Pro Thr Phe Leu Arg Glu Leu Ser Asp Glu Thr Val Val Leu Gly Gln 1105 1110 1115 1120 Ser Val Thr Leu Ala Cys Gln Val Ser Ala Gln Pro Ala Ala Gln Ala 1125 1130 1135 Thr Trp Ser Lys Asp Gly Ala Pro Leu Glu Ser Ser Ser Arg Val Leu 1140 1145 1150 Ile Ser Ala Thr Leu Lys Asn Phe Gln Leu Leu Thr Ile Leu Val Val 1155 1160 1165 Val Ala Glu Asp Leu Gly Val Tyr Thr Cys Ser Val Ser Asn Ala Leu 1170 1175 1180 Gly Thr Val Thr Thr Thr Gly Val Leu Arg Lys Ala Glu Arg Pro Ser 1185 1190 1195 1200 Ser Ser Pro Cys Pro Asp Ile Gly Glu Val Tyr Ala Asp Gly Val Leu 1205 1210 1215 Leu Val Trp Lys Pro Val Glu Ser Tyr Gly Pro Val Thr Tyr Ile Val 1220 1225 1230 Gln Cys Ser Leu Glu Gly Gly Ser Trp Thr Thr Leu Ala Ser Asp Ile 1235 1240 1245 Phe Asp Cys Cys Tyr Leu Thr Ser Lys Leu Ser Arg Gly Gly Thr Tyr 1250 1255 1260 Thr Phe Arg Thr Ala Cys Val Ser Lys Ala Gly Met Gly Pro Tyr Ser 1265 1270 1275 1280 Ser Pro Ser Glu Gln Val Leu Leu Gly Ala Pro Ser His Leu Ala Ser 1285 1290 1295 Glu Glu Glu Ser Gln Gly Arg Ser Ala Gln Pro Leu Pro Ser Thr Lys 1300 1305 1310 Thr Phe Ala Phe Gln Thr Gln Ile Gln Arg Gly Arg Phe Ser Val Val 1315 1320 1325 Arg Gln Cys Trp Glu Lys Ala Ser Gly Arg Ala Leu Ala Ala Lys Ile 1330 1335 1340 Ile Pro Tyr His Pro Lys Asp Lys Thr Ala Val Leu Arg Glu Tyr Glu 1345 1350 1355 1360 Ala Leu Lys Gly Leu Arg His Pro His Leu Ala Gln Leu His Ala Ala 1365 1370 1375 Tyr Leu Ser Pro Arg His Leu Val Leu Ile Leu Glu Leu Cys Ser Gly 1380 1385 1390 Pro Glu Leu Leu Pro Cys Leu Ala Glu Arg Ala Ser Tyr Ser Glu Ser 1395 1400 1405 Glu Val Lys Asp Tyr Leu Trp Gln Met Leu Ser Ala Thr Gln Tyr Leu 1410 1415 1420 His Asn Gln His Ile Leu His Leu Asp Leu Arg Ser Glu Asn Met Ile 1425 1430 1435 1440 Ile Thr Glu Tyr Asn Leu Leu Lys Val Val Asp Leu Gly Asn Ala Gln 1445 1450 1455 Ser Leu Ser Gln Glu Lys Val Leu Pro Ser Asp Lys Phe Lys Asp Tyr 1460 1465 1470 Leu Glu Thr Met Ala Pro Glu Leu Leu Glu Gly Gln Gly Ala Val Pro 1475 1480 1485 Gln Thr Asp Ile Trp Ala Ile Gly Val Thr Ala Phe Ile Met Leu Ser 1490 1495 1500 Ala Glu Tyr Pro Val Ser Ser Glu Gly Ala Arg Asp Leu Gln Arg Gly 1505 1510 1515 1520 Leu Arg Lys Gly Leu Val Arg Leu Ser Arg Cys Tyr Ala Gly Leu Ser 1525 1530 1535 Gly Gly Ala Val Ala Phe Leu Arg Ser Thr Leu Cys Ala Gln Pro Trp 1540 1545 1550 Gly Arg Pro Cys Ala Ser Ser Cys Leu Gln Cys Pro Trp Leu Thr Glu 1555 1560 1565 Glu Gly Pro Ala Cys Ser Arg Pro Ala Pro Val Thr Phe Pro Thr Ala 1570 1575 1580 Arg Leu Arg Val Phe Val Arg Asn Arg Glu Lys Arg Arg Ala Leu Leu 1585 1590 1595 1600 Tyr Lys Arg His Asn Leu Ala Gln Val Arg 1605 1610 5 7928 DNA Homo sapiens CDS (60)...(7847) 5 gaattcggcg cgccagatat cacacgtgcc aaggggctgg ctcactggtg ccgccccga 59 atg ctg gag agg ttc acc ccc aag aaa gtg aag aaa ggc tcc agc atc 107 Met Leu Glu Arg Phe Thr Pro Lys Lys Val Lys Lys Gly Ser Ser Ile 1 5 10 15 acc ttc tct gtg aag gta gaa gga cgc ccg gtg ccc acc gtg cac tgg 155 Thr Phe Ser Val Lys Val Glu Gly Arg Pro Val Pro Thr Val His Trp 20 25 30 ctc agg gag gag gct gag aga ggc gtg ctg tgg att ggc cct gac aca 203 Leu Arg Glu Glu Ala Glu Arg Gly Val Leu Trp Ile Gly Pro Asp Thr 35 40 45 ccg ggc tac acc gtg gcc agc tct gcg cag cag cac agc ctg gtc ctg 251 Pro Gly Tyr Thr Val Ala Ser Ser Ala Gln Gln His Ser Leu Val Leu 50 55 60 ctg gac gtg ggc cgg cag cac cag ggc acc tac aca tgc att gcc agc 299 Leu Asp Val Gly Arg Gln His Gln Gly Thr Tyr Thr Cys Ile Ala Ser 65 70 75 80 aac gct gcc ggc cag gcc ctc tgc tcc gcc agc ctg cac gtc tcg ggc 347 Asn Ala Ala Gly Gln Ala Leu Cys Ser Ala Ser Leu His Val Ser Gly 85 90 95 ctg cct aag gtg gag gag cag gag aaa gtg aag gaa gcg ctg att tcc 395 Leu Pro Lys Val Glu Glu Gln Glu Lys Val Lys Glu Ala Leu Ile Ser 100 105 110 act ttc ctg cag ggg acc aca caa gcc atc tca gca cag ggg ttg gaa 443 Thr Phe Leu Gln Gly Thr Thr Gln Ala Ile Ser Ala Gln Gly Leu Glu 115 120 125 act gcg agt ttt gct gac ctt ggt ggg cag agg aaa gaa gag cct ctg 491 Thr Ala Ser Phe Ala Asp Leu Gly Gly Gln Arg Lys Glu Glu Pro Leu 130 135 140 gct gcc aag gag gcc ctc ggc cac ctg tcc ctc gct gag gtg ggc aca 539 Ala Ala Lys Glu Ala Leu Gly His Leu Ser Leu Ala Glu Val Gly Thr 145 150 155 160 gag gag ttc ctg cag aaa ctg acc tcc cag atc act gag atg gta tcg 587 Glu Glu Phe Leu Gln Lys Leu Thr Ser Gln Ile Thr Glu Met Val Ser 165 170 175 gcc aag atc acg cag gcc aag ctg cag gtg cca gga ggt gac agt gat 635 Ala Lys Ile Thr Gln Ala Lys Leu Gln Val Pro Gly Gly Asp Ser Asp 180 185 190 gag gac tcc aag aca cca tct gca tcc ccc cgc cat ggc cga tca cgg 683 Glu Asp Ser Lys Thr Pro Ser Ala Ser Pro Arg His Gly Arg Ser Arg 195 200 205 cca tcc tcc agc atc cag gag tct tcc tca gag tca gag gac ggc gat 731 Pro Ser Ser Ser Ile Gln Glu Ser Ser Ser Glu Ser Glu Asp Gly Asp 210 215 220 gcc cga ggc gag atc ttt gac atc tac gtg gtc acc gct gac tac ctg 779 Ala Arg Gly Glu Ile Phe Asp Ile Tyr Val Val Thr Ala Asp Tyr Leu 225 230 235 240 ccc cta ggg gct gag cag gat gcc atc acg ctg cgg gaa ggc cag tat 827 Pro Leu Gly Ala Glu Gln Asp Ala Ile Thr Leu Arg Glu Gly Gln Tyr 245 250 255 gtg gag gtc ctg gat gca gcc cac cca ctg cgc tgg ctt gtc cgc acc 875 Val Glu Val Leu Asp Ala Ala His Pro Leu Arg Trp Leu Val Arg Thr 260 265 270 aag ccc acc aag tcc agc ccc tca cgg cag ggc tgg gtg tca cca gcc 923 Lys Pro Thr Lys Ser Ser Pro Ser Arg Gln Gly Trp Val Ser Pro Ala 275 280 285 tac ctg gac agg agg ctc aag ctg tca cct gag tgg ggg gcc gct gag 971 Tyr Leu Asp Arg Arg Leu Lys Leu Ser Pro Glu Trp Gly Ala Ala Glu 290 295 300 gcc cct gag ttc cct ggg gag gct gtg tct gaa gac gaa tac aag gca 1019 Ala Pro Glu Phe Pro Gly Glu Ala Val Ser Glu Asp Glu Tyr Lys Ala 305 310 315 320 agg ctg agc tct gtg atc cag gag ctg ctg agt tct gag cag gcc ttc 1067 Arg Leu Ser Ser Val Ile Gln Glu Leu Leu Ser Ser Glu Gln Ala Phe 325 330 335 gtg gag gag ctg cag ttc ctg cag agc cac cac ctg cag cac ctg gag 1115 Val Glu Glu Leu Gln Phe Leu Gln Ser His His Leu Gln His Leu Glu 340 345 350 cgc tgc ccc cac gtg ccc ata gcc gtg gcc ggc cag aag gca gtc atc 1163 Arg Cys Pro His Val Pro Ile Ala Val Ala Gly Gln Lys Ala Val Ile 355 360 365 ttc cgc aat gtg cgg gac atc ggc cgc ttc cac agc agc ttc ctg cag 1211 Phe Arg Asn Val Arg Asp Ile Gly Arg Phe His Ser Ser Phe Leu Gln 370 375 380 gag ttg cag cag tgc gac acg gac gac gac gtg gcc atg tgc ttc atc 1259 Glu Leu Gln Gln Cys Asp Thr Asp Asp Asp Val Ala Met Cys Phe Ile 385 390 395 400 aag aac cag gcg gcc ttt gag cag tac ctg gag ttc ctg gtg ggg cgt 1307 Lys Asn Gln Ala Ala Phe Glu Gln Tyr Leu Glu Phe Leu Val Gly Arg 405 410 415 gtg cag gct gag tcg gtg gtc gtc agc acg gcc atc cag gag ttc tac 1355 Val Gln Ala Glu Ser Val Val Val Ser Thr Ala Ile Gln Glu Phe Tyr 420 425 430 aag aaa tac gcg gag gag gcc ctg ttg gca ggg gac ccc tct cag ccc 1403 Lys Lys Tyr Ala Glu Glu Ala Leu Leu Ala Gly Asp Pro Ser Gln Pro 435 440 445 ccg cca cca cct ctg cag cac tac ctg gag cag cca gtg gag cgg gtg 1451 Pro Pro Pro Pro Leu Gln His Tyr Leu Glu Gln Pro Val Glu Arg Val 450 455 460 cag cgc tac cag gcc ttg ctg aag gag ttg atc cgc aac aag gcg cgg 1499 Gln Arg Tyr Gln Ala Leu Leu Lys Glu Leu Ile Arg Asn Lys Ala Arg 465 470 475 480 aac aga cag aac tgc gcg ctg ctg gag cag gcc tat gcc gtg gtg tct 1547 Asn Arg Gln Asn Cys Ala Leu Leu Glu Gln Ala Tyr Ala Val Val Ser 485 490 495 gcc ctg cca cag cgc gct gag aac aag ctg cac gtg tcc ctc atg gag 1595 Ala Leu Pro Gln Arg Ala Glu Asn Lys Leu His Val Ser Leu Met Glu 500 505 510 aac tac cca ggc acc ctg gag gcc ctg ggc gag ccc atc cgc cag ggc 1643 Asn Tyr Pro Gly Thr Leu Glu Ala Leu Gly Glu Pro Ile Arg Gln Gly 515 520 525 cac ttc atc gtg tgg gag ggt gca ccg ggg gcc cgc atg ccc tgg aag 1691 His Phe Ile Val Trp Glu Gly Ala Pro Gly Ala Arg Met Pro Trp Lys 530 535 540 ggc cac aac cgt cac gtg ttc ctc ttc cgc aac cac ctg gta atc tgc 1739 Gly His Asn Arg His Val Phe Leu Phe Arg Asn His Leu Val Ile Cys 545 550 555 560 aag ccc cgg cga gac tcc cgc acc gat acc gtc agc tac gtg ttc cgg 1787 Lys Pro Arg Arg Asp Ser Arg Thr Asp Thr Val Ser Tyr Val Phe Arg 565 570 575 aac atg atg aag ctg agc agc atc gac ctg aac gac cag gtg gag ggg 1835 Asn Met Met Lys Leu Ser Ser Ile Asp Leu Asn Asp Gln Val Glu Gly 580 585 590 gat gac cgc gcc ttc gag gtg tgg cag gag cgg gag gac tcg gtg cgc 1883 Asp Asp Arg Ala Phe Glu Val Trp Gln Glu Arg Glu Asp Ser Val Arg 595 600 605 aag tac ctg ctg cag gca cgg aca gcc att atc aag agc tcg tgg gtg 1931 Lys Tyr Leu Leu Gln Ala Arg Thr Ala Ile Ile Lys Ser Ser Trp Val 610 615 620 aag gag atc tgt ggc atc cag cag cgt ctg gcc ctg cct gtg tgg cgg 1979 Lys Glu Ile Cys Gly Ile Gln Gln Arg Leu Ala Leu Pro Val Trp Arg 625 630 635 640 ccc ccg gac ttt gaa gag gag ctg gcc gac tgc aca gcc gag ctg ggt 2027 Pro Pro Asp Phe Glu Glu Glu Leu Ala Asp Cys Thr Ala Glu Leu Gly 645 650 655 gag aca gtc aag ctg gcc tgc cgc gtg acg ggc aca ccc aag cct gtc 2075 Glu Thr Val Lys Leu Ala Cys Arg Val Thr Gly Thr Pro Lys Pro Val 660 665 670 atc agc tgg tac aaa gat ggg aaa gca gtg cag gtg gac ccc cac cac 2123 Ile Ser Trp Tyr Lys Asp Gly Lys Ala Val Gln Val Asp Pro His His 675 680 685 atc ctc att gaa gac cct gat ggc tcg tgt gca ctc atc ctg gac agc 2171 Ile Leu Ile Glu Asp Pro Asp Gly Ser Cys Ala Leu Ile Leu Asp Ser 690 695 700 ctg acc ggt gtg gac tct ggc cag tac atg tgc ttc gcg gcc agc gcc 2219 Leu Thr Gly Val Asp Ser Gly Gln Tyr Met Cys Phe Ala Ala Ser Ala 705 710 715 720 gct ggc aac tgc agt acc ctg ggc aag atc ctg gtg caa gtc cca cca 2267 Ala Gly Asn Cys Ser Thr Leu Gly Lys Ile Leu Val Gln Val Pro Pro 725 730 735 cgg ttc gtg aac aag gtc cgg gcc tca ccc ttt gtg gag gga gag gac 2315 Arg Phe Val Asn Lys Val Arg Ala Ser Pro Phe Val Glu Gly Glu Asp 740 745 750 gcc cag ttc acc tgc acc atc gaa ggc gcc ccg tac ccg cag atc agg 2363 Ala Gln Phe Thr Cys Thr Ile Glu Gly Ala Pro Tyr Pro Gln Ile Arg 755 760 765 tgg tac aag gac ggg gcc ctg ctg acc act ggc aac aag ttc cag aca 2411 Trp Tyr Lys Asp Gly Ala Leu Leu Thr Thr Gly Asn Lys Phe Gln Thr 770 775 780 ctg agt gag cct cgc agc ggc ctg cta gtg ctg gtg atc cgg gcg gcc 2459 Leu Ser Glu Pro Arg Ser Gly Leu Leu Val Leu Val Ile Arg Ala Ala 785 790 795 800 agc aag gag gac ctg ggg ctc tac gag tgt gag ctg gtg aac cgg ctg 2507 Ser Lys Glu Asp Leu Gly Leu Tyr Glu Cys Glu Leu Val Asn Arg Leu 805 810 815 ggc tcc gcg cgg gct agt gcg gag ctg cgc att cag agc ccc atg ctg 2555 Gly Ser Ala Arg Ala Ser Ala Glu Leu Arg Ile Gln Ser Pro Met Leu 820 825 830 cag gcc cag gag cag tgt cac agg gag cag ctc gtg gct gca gtg gaa 2603 Gln Ala Gln Glu Gln Cys His Arg Glu Gln Leu Val Ala Ala Val Glu 835 840 845 gac acc acc ctg gag cga gcg gac cag gag gtc aca tct gtc ctg aag 2651 Asp Thr Thr Leu Glu Arg Ala Asp Gln Glu Val Thr Ser Val Leu Lys 850 855 860 aga ctg ctg ggc ccc aag gcg cca ggc ccc tcc aca ggg gac ctc act 2699 Arg Leu Leu Gly Pro Lys Ala Pro Gly Pro Ser Thr Gly Asp Leu Thr 865 870 875 880 ggc cct ggc ccc tgc ccc agg ggg gca ccc gca ctc cag gaa acc ggc 2747 Gly Pro Gly Pro Cys Pro Arg Gly Ala Pro Ala Leu Gln Glu Thr Gly 885 890 895 tcc cag ccc cca gtc acc gga act tcg gag gca cct gcc gtg ccc ccg 2795 Ser Gln Pro Pro Val Thr Gly Thr Ser Glu Ala Pro Ala Val Pro Pro 900 905 910 agg gtg cca cag ccc ctc ctc cac gaa ggc cca gag cag gag ccg gag 2843 Arg Val Pro Gln Pro Leu Leu His Glu Gly Pro Glu Gln Glu Pro Glu 915 920 925 gcc att gcc aga gcc cag gaa tgg act gtg ccc att cgg atg gag ggt 2891 Ala Ile Ala Arg Ala Gln Glu Trp Thr Val Pro Ile Arg Met Glu Gly 930 935 940 gca gcc tgg ccc ggg gca ggc aca ggg gag ctg ctc tgg gac gtc cac 2939 Ala Ala Trp Pro Gly Ala Gly Thr Gly Glu Leu Leu Trp Asp Val His 945 950 955 960 agc cac gtg gtc aga gag acc aca cag agg acc tac aca tac cag gcc 2987 Ser His Val Val Arg Glu Thr Thr Gln Arg Thr Tyr Thr Tyr Gln Ala 965 970 975 atc gac acg cac acc gca cgg ccc cca tcc atg cag gta acc atc gag 3035 Ile Asp Thr His Thr Ala Arg Pro Pro Ser Met Gln Val Thr Ile Glu 980 985 990 gat gtg cag gca cag aca ggc gga acg gcc caa ttc gag gct atc att 3083 Asp Val Gln Ala Gln Thr Gly Gly Thr Ala Gln Phe Glu Ala Ile Ile 995 1000 1005 gag ggc gac cca cag ccc tcg gtg acc tgg tac aag gac agc gtc cag 3131 Glu Gly Asp Pro Gln Pro Ser Val Thr Trp Tyr Lys Asp Ser Val Gln 1010 1015 1020 ctg gtg gac agc acc cgg ctt agc cag cag caa gaa ggc acc aca tac 3179 Leu Val Asp Ser Thr Arg Leu Ser Gln Gln Gln Glu Gly Thr Thr Tyr 1025 1030 1035 1040 tcc ctg gtg ctg agg cat gtg gcc tcg aag gat gcc ggc gtt tac acc 3227 Ser Leu Val Leu Arg His Val Ala Ser Lys Asp Ala Gly Val Tyr Thr 1045 1050 1055 tgc ctg gcc caa aac act ggt ggc cag gtg ctc tgc aag gca gag ctg 3275 Cys Leu Ala Gln Asn Thr Gly Gly Gln Val Leu Cys Lys Ala Glu Leu 1060 1065 1070 ctg gtg ctt ggg ggg gac aat gag ccg gac tca gag aag caa agc cac 3323 Leu Val Leu Gly Gly Asp Asn Glu Pro Asp Ser Glu Lys Gln Ser His 1075 1080 1085 cgg agg aag ctg cac tcc ttc tat gag gtc aag gag gag att gga agg 3371 Arg Arg Lys Leu His Ser Phe Tyr Glu Val Lys Glu Glu Ile Gly Arg 1090 1095 1100 ggc gtg ttt ggc ttc gta aaa aga gtg cag cac aaa gga aac aag atc 3419 Gly Val Phe Gly Phe Val Lys Arg Val Gln His Lys Gly Asn Lys Ile 1105 1110 1115 1120 ttg tgc gct gcc aag ttc atc ccc cta cgg agc aga act cgg gcc cag 3467 Leu Cys Ala Ala Lys Phe Ile Pro Leu Arg Ser Arg Thr Arg Ala Gln 1125 1130 1135 gca tac agg gag cga gac atc ctg gcc gcg ctg agc cac ccg ctg gtc 3515 Ala Tyr Arg Glu Arg Asp Ile Leu Ala Ala Leu Ser His Pro Leu Val 1140 1145 1150 acg ggg ctg ctg gac cag ttt gag acc cgc aag acc ctc atc ctc atc 3563 Thr Gly Leu Leu Asp Gln Phe Glu Thr Arg Lys Thr Leu Ile Leu Ile 1155 1160 1165 ctg gag ctg tgc tca tcc gag gag ctg ctg gac cgc ctg tac agg aag 3611 Leu Glu Leu Cys Ser Ser Glu Glu Leu Leu Asp Arg Leu Tyr Arg Lys 1170 1175 1180 ggc gtg gtg acg gag gcc gag gtc aag gtc tac atc cag cag ctg gtg 3659 Gly Val Val Thr Glu Ala Glu Val Lys Val Tyr Ile Gln Gln Leu Val 1185 1190 1195 1200 gag ggg ctg cac tac ctg cac agc cat ggc gtt ctc cac ctg gac ata 3707 Glu Gly Leu His Tyr Leu His Ser His Gly Val Leu His Leu Asp Ile 1205 1210 1215 aag ccc tct aac atc ctg atg gtg cat cct gcc cgg gaa gac att aaa 3755 Lys Pro Ser Asn Ile Leu Met Val His Pro Ala Arg Glu Asp Ile Lys 1220 1225 1230 atc tgc gac ttt ggc ttt gcc cag aac atc acc cca gca gag ctg cag 3803 Ile Cys Asp Phe Gly Phe Ala Gln Asn Ile Thr Pro Ala Glu Leu Gln 1235 1240 1245 ttc agc cag tac ggc tcc cct gag ttc gtc tcc ccc gag atc atc cag 3851 Phe Ser Gln Tyr Gly Ser Pro Glu Phe Val Ser Pro Glu Ile Ile Gln 1250 1255 1260 cag aac cct gtg agc gaa gcc tcc gac att tgg gcc atg ggt gtc atc 3899 Gln Asn Pro Val Ser Glu Ala Ser Asp Ile Trp Ala Met Gly Val Ile 1265 1270 1275 1280 tcc tac ctc agc ctg acc tgc tca tcc cca ttt gcc ggc gag agt gac 3947 Ser Tyr Leu Ser Leu Thr Cys Ser Ser Pro Phe Ala Gly Glu Ser Asp 1285 1290 1295 cgt gcc acc ctc ctg aac gtc ctg gag ggg cgc gtg tca tgg agc agc 3995 Arg Ala Thr Leu Leu Asn Val Leu Glu Gly Arg Val Ser Trp Ser Ser 1300 1305 1310 ccc atg gct gcc cac ctc agc gaa gac gcc aaa gac ttc atc aag gct 4043 Pro Met Ala Ala His Leu Ser Glu Asp Ala Lys Asp Phe Ile Lys Ala 1315 1320 1325 acg ctg cag aga gcc cct cag gcc cgg cct agt gcg gcc cag tgc ctc 4091 Thr Leu Gln Arg Ala Pro Gln Ala Arg Pro Ser Ala Ala Gln Cys Leu 1330 1335 1340 tcc cac ccc tgg ttc ctg aaa tcc atg cct gcg gag gag gcc cac ttc 4139 Ser His Pro Trp Phe Leu Lys Ser Met Pro Ala Glu Glu Ala His Phe 1345 1350 1355 1360 atc aac acc aag cag ctc aag ttc ctc ctg gcc cga agt cgc tgg cag 4187 Ile Asn Thr Lys Gln Leu Lys Phe Leu Leu Ala Arg Ser Arg Trp Gln 1365 1370 1375 cgt tcc ctg atg agc tac aag tcc atc ctg gtg atg cgc tcc atc cct 4235 Arg Ser Leu Met Ser Tyr Lys Ser Ile Leu Val Met Arg Ser Ile Pro 1380 1385 1390 gag ctg ctg cgg ggc cca ccc gac agc ccc tcc ctc ggc gta gcc cgg 4283 Glu Leu Leu Arg Gly Pro Pro Asp Ser Pro Ser Leu Gly Val Ala Arg 1395 1400 1405 cac ctc tgc agg gac act ggt ggc tcc tcc agt tcc tcc tcc tcc tct 4331 His Leu Cys Arg Asp Thr Gly Gly Ser Ser Ser Ser Ser Ser Ser Ser 1410 1415 1420 gac aac gag ctc gcc cca ttt gcc cgg gct aag tca ctg cca ccc tcc 4379 Asp Asn Glu Leu Ala Pro Phe Ala Arg Ala Lys Ser Leu Pro Pro Ser 1425 1430 1435 1440 ccg gtg aca cac tca cca ctg ctg cac ccc cgg ggc ttc ctg cgg ccc 4427 Pro Val Thr His Ser Pro Leu Leu His Pro Arg Gly Phe Leu Arg Pro 1445 1450 1455 tcg gcc agc ctg cct gag gaa gcc gag gcc agt gag cgc tcc acc gag 4475 Ser Ala Ser Leu Pro Glu Glu Ala Glu Ala Ser Glu Arg Ser Thr Glu 1460 1465 1470 gcc cca gct ccg cct gca tct ccc gag ggt gcc ggg cca ccg gcc gcc 4523 Ala Pro Ala Pro Pro Ala Ser Pro Glu Gly Ala Gly Pro Pro Ala Ala 1475 1480 1485 cag ggc tgc gtg ccc cgg cac agc gtc atc cgc agc ctg ttc tac cac 4571 Gln Gly Cys Val Pro Arg His Ser Val Ile Arg Ser Leu Phe Tyr His 1490 1495 1500 cag gcg ggt gag agc cct gag cac ggg gcc ctg gcc ccg ggg agc agg 4619 Gln Ala Gly Glu Ser Pro Glu His Gly Ala Leu Ala Pro Gly Ser Arg 1505 1510 1515 1520 cgg cac ccg gcc cgg cgg cgg cac ctg ctg aag ggc ggg tac att gcg 4667 Arg His Pro Ala Arg Arg Arg His Leu Leu Lys Gly Gly Tyr Ile Ala 1525 1530 1535 ggg gcg ctg cca ggc ctg cgc gag cca ctg atg gag cac cgc gtg ctg 4715 Gly Ala Leu Pro Gly Leu Arg Glu Pro Leu Met Glu His Arg Val Leu 1540 1545 1550 gag gag gag gcc gcc agg gag gag cag gcc acc ctc ctg gcc aaa gcc 4763 Glu Glu Glu Ala Ala Arg Glu Glu Gln Ala Thr Leu Leu Ala Lys Ala 1555 1560 1565 ccc tca ttc gag act gcc ctc cgg ctg cct gcc tct ggc acc cac ttg 4811 Pro Ser Phe Glu Thr Ala Leu Arg Leu Pro Ala Ser Gly Thr His Leu 1570 1575 1580 gcc cct ggc cac agc cac tcc ctg gaa cat gac tct ccg agc acc ccc 4859 Ala Pro Gly His Ser His Ser Leu Glu His Asp Ser Pro Ser Thr Pro 1585 1590 1595 1600 cgc ccc tcc tcg gag gcc tgc ggt gag gca cag cga ctg cct tca gcc 4907 Arg Pro Ser Ser Glu Ala Cys Gly Glu Ala Gln Arg Leu Pro Ser Ala 1605 1610 1615 ccc tcc ggg ggg gcc cct atc agg gac atg ggg cac cct cag ggc tcc 4955 Pro Ser Gly Gly Ala Pro Ile Arg Asp Met Gly His Pro Gln Gly Ser 1620 1625 1630 aag cag ctt cca tcc act ggt ggc cac cca ggc act gct cag cca gag 5003 Lys Gln Leu Pro Ser Thr Gly Gly His Pro Gly Thr Ala Gln Pro Glu 1635 1640 1645 agg cca tcc ccg gac agc cct tgg ggg cag cca gcc cct ttc tgc cac 5051 Arg Pro Ser Pro Asp Ser Pro Trp Gly Gln Pro Ala Pro Phe Cys His 1650 1655 1660 ccc aag cag ggt tct gcc ccc cag gag ggc tgc agc ccc cac cca gca 5099 Pro Lys Gln Gly Ser Ala Pro Gln Glu Gly Cys Ser Pro His Pro Ala 1665 1670 1675 1680 gtt gcc cca tgc cct cct ggc tcc ttc cct cca gga tct tgc aaa gag 5147 Val Ala Pro Cys Pro Pro Gly Ser Phe Pro Pro Gly Ser Cys Lys Glu 1685 1690 1695 gcc ccc tta gta ccc tca agc ccc ttc ttg gga cag ccc cag gca ccc 5195 Ala Pro Leu Val Pro Ser Ser Pro Phe Leu Gly Gln Pro Gln Ala Pro 1700 1705 1710 cct gcc cct gcc aaa gca agc ccc cca ttg gac tct aag atg ggg cct 5243 Pro Ala Pro Ala Lys Ala Ser Pro Pro Leu Asp Ser Lys Met Gly Pro 1715 1720 1725 gga gac atc tct ctt cct ggg agg cca aaa ccc ggc ccc tgc agt tcc 5291 Gly Asp Ile Ser Leu Pro Gly Arg Pro Lys Pro Gly Pro Cys Ser Ser 1730 1735 1740 cca ggg tca gcc tcc cag gcg agc tct tcc caa gtg agc tcc ctc agg 5339 Pro Gly Ser Ala Ser Gln Ala Ser Ser Ser Gln Val Ser Ser Leu Arg 1745 1750 1755 1760 gtg ggc tcc tcc cag gtg ggc aca gag cct ggc ccc tcc ctg gat gcg 5387 Val Gly Ser Ser Gln Val Gly Thr Glu Pro Gly Pro Ser Leu Asp Ala 1765 1770 1775 gag ggc tgg acc cag gag gct gag gat ctg tcc gac tcc aca ccc acc 5435 Glu Gly Trp Thr Gln Glu Ala Glu Asp Leu Ser Asp Ser Thr Pro Thr 1780 1785 1790 ttg cag cgg cct cag gaa cag gcg acc atg cgc aag ttc tcc ctg ggt 5483 Leu Gln Arg Pro Gln Glu Gln Ala Thr Met Arg Lys Phe Ser Leu Gly 1795 1800 1805 ggt cgc ggg ggc tac gca ggc gtg gct ggc tat ggc acc ttt gcc ttt 5531 Gly Arg Gly Gly Tyr Ala Gly Val Ala Gly Tyr Gly Thr Phe Ala Phe 1810 1815 1820 ggt gga gat gca ggg ggc atg ctg ggg cag ggg ccc atg tgg gcc agg 5579 Gly Gly Asp Ala Gly Gly Met Leu Gly Gln Gly Pro Met Trp Ala Arg 1825 1830 1835 1840 ata gcc tgg gct gtg tcc cag tca gag gag gag gag cag gag gag gcc 5627 Ile Ala Trp Ala Val Ser Gln Ser Glu Glu Glu Glu Gln Glu Glu Ala 1845 1850 1855 agg gct gag tcc cag tcg gag gag cag cag gag gcc agg gct gag agc 5675 Arg Ala Glu Ser Gln Ser Glu Glu Gln Gln Glu Ala Arg Ala Glu Ser 1860 1865 1870 cca ctg ccc cag gtc agt gca agg cct gtg cct gag gtc ggc agg gct 5723 Pro Leu Pro Gln Val Ser Ala Arg Pro Val Pro Glu Val Gly Arg Ala 1875 1880 1885 ccc acc agg agc tct cca gag ccc acc cca tgg gag gac atc ggg cag 5771 Pro Thr Arg Ser Ser Pro Glu Pro Thr Pro Trp Glu Asp Ile Gly Gln 1890 1895 1900 gtc tcc ctg gtg cag atc cgg gac ctg tca ggt gat gcg gag gcg gcc 5819 Val Ser Leu Val Gln Ile Arg Asp Leu Ser Gly Asp Ala Glu Ala Ala 1905 1910 1915 1920 gac aca ata tcc ctg gac att tcc gag gtg gac ccc gcc tac ctc aac 5867 Asp Thr Ile Ser Leu Asp Ile Ser Glu Val Asp Pro Ala Tyr Leu Asn 1925 1930 1935 ctc tca gac ctg tac gat atc aag tac ctc cca ttc gag ttt atg atc 5915 Leu Ser Asp Leu Tyr Asp Ile Lys Tyr Leu Pro Phe Glu Phe Met Ile 1940 1945 1950 ttc agg aaa gtc ccc aag tcc gct cag cca gag ccg ccc tcc ccc atg 5963 Phe Arg Lys Val Pro Lys Ser Ala Gln Pro Glu Pro Pro Ser Pro Met 1955 1960 1965 gct gag gag gag ctg gcc gag ttc ccg gag ccc acg tgg ccc tgg cca 6011 Ala Glu Glu Glu Leu Ala Glu Phe Pro Glu Pro Thr Trp Pro Trp Pro 1970 1975 1980 ggt gaa ctg ggc ccc cac gca ggc ctg gag atc aca gag gag tca gag 6059 Gly Glu Leu Gly Pro His Ala Gly Leu Glu Ile Thr Glu Glu Ser Glu 1985 1990 1995 2000 gat gtg gac gcg ctg ctg gca gag gct gcc gtg ggc agg aag cgc aag 6107 Asp Val Asp Ala Leu Leu Ala Glu Ala Ala Val Gly Arg Lys Arg Lys 2005 2010 2015 tgg tcc tcg ccg tca cgc agc ctc ttc cac ttc cct ggg agg cac ctg 6155 Trp Ser Ser Pro Ser Arg Ser Leu Phe His Phe Pro Gly Arg His Leu 2020 2025 2030 ccg ctg gat gag cct gca gag ctg ggg ctg cgt gag aga gtg aag gcc 6203 Pro Leu Asp Glu Pro Ala Glu Leu Gly Leu Arg Glu Arg Val Lys Ala 2035 2040 2045 tcc gtg gag cac atc tcc cgg atc ctg aag ggc agg ccg gaa ggt ctg 6251 Ser Val Glu His Ile Ser Arg Ile Leu Lys Gly Arg Pro Glu Gly Leu 2050 2055 2060 gag aag gag ggg ccc ccc agg aag aag cca ggc ctt gct tcc ttc cgg 6299 Glu Lys Glu Gly Pro Pro Arg Lys Lys Pro Gly Leu Ala Ser Phe Arg 2065 2070 2075 2080 ctc tca ggt ctg aag agc tgg gac cga gcg ccg aca ttc cta agg gag 6347 Leu Ser Gly Leu Lys Ser Trp Asp Arg Ala Pro Thr Phe Leu Arg Glu 2085 2090 2095 ctc tca gat gag act gtg gtc ctg ggc cag tca gtg aca ctg gcc tgc 6395 Leu Ser Asp Glu Thr Val Val Leu Gly Gln Ser Val Thr Leu Ala Cys 2100 2105 2110 cag gtg tca gcc cag cca gct gcc cag gcc acc tgg agc aaa gac gga 6443 Gln Val Ser Ala Gln Pro Ala Ala Gln Ala Thr Trp Ser Lys Asp Gly 2115 2120 2125 gcc ccc ctg gag agc agc agc cgt gtc ctc atc tct gcc acc ctc aag 6491 Ala Pro Leu Glu Ser Ser Ser Arg Val Leu Ile Ser Ala Thr Leu Lys 2130 2135 2140 aac ttc cag ctt ctg acc atc ctg gtg gtg gtg gct gag gac ctg ggt 6539 Asn Phe Gln Leu Leu Thr Ile Leu Val Val Val Ala Glu Asp Leu Gly 2145 2150 2155 2160 gtg tac acc tgc agc gtg agc aat gcg ctg ggg aca gtg acc acc acg 6587 Val Tyr Thr Cys Ser Val Ser Asn Ala Leu Gly Thr Val Thr Thr Thr 2165 2170 2175 ggc gtc ctc cgg aag gca gag cgc ccc tca tct tcg cca tgc ccg gat 6635 Gly Val Leu Arg Lys Ala Glu Arg Pro Ser Ser Ser Pro Cys Pro Asp 2180 2185 2190 atc ggg gag gtg tac gcg gat ggg gtg ctg ctg gtc tgg aag ccc gtg 6683 Ile Gly Glu Val Tyr Ala Asp Gly Val Leu Leu Val Trp Lys Pro Val 2195 2200 2205 gaa tcc tac ggc cct gtg acc tac att gtg cag tgc agc cta gaa ggc 6731 Glu Ser Tyr Gly Pro Val Thr Tyr Ile Val Gln Cys Ser Leu Glu Gly 2210 2215 2220 ggc agc tgg acc aca ctg gcc tcc gac atc ttt gac tgc tgc tac ctg 6779 Gly Ser Trp Thr Thr Leu Ala Ser Asp Ile Phe Asp Cys Cys Tyr Leu 2225 2230 2235 2240 acc agc aag ctc tcc cgg ggt ggc acc tac acc ttc cgc acg gca tgt 6827 Thr Ser Lys Leu Ser Arg Gly Gly Thr Tyr Thr Phe Arg Thr Ala Cys 2245 2250 2255 gtc agc aag gca gga atg ggt ccc tac agc agc ccc tcg gag caa gtc 6875 Val Ser Lys Ala Gly Met Gly Pro Tyr Ser Ser Pro Ser Glu Gln Val 2260 2265 2270 ctc ctg gga gcg ccc agc cac ctg gcc tct gag gag gag agc cag ggg 6923 Leu Leu Gly Ala Pro Ser His Leu Ala Ser Glu Glu Glu Ser Gln Gly 2275 2280 2285 cgg tca gcc caa ccc ctg ccc agc aca aag acc ttc gca ttc cag aca 6971 Arg Ser Ala Gln Pro Leu Pro Ser Thr Lys Thr Phe Ala Phe Gln Thr 2290 2295 2300 cag atc cag agg ggc cgc ttc agc gtg gtg cgg caa tgc tgg gag aag 7019 Gln Ile Gln Arg Gly Arg Phe Ser Val Val Arg Gln Cys Trp Glu Lys 2305 2310 2315 2320 gcc agc ggg cgg gcg ctg gcc gcc aag atc atc ccc tac cac ccc aag 7067 Ala Ser Gly Arg Ala Leu Ala Ala Lys Ile Ile Pro Tyr His Pro Lys 2325 2330 2335 gac aag aca gca gtg ctg cgc gaa tac gag gcc ctc aag ggc ctg cgc 7115 Asp Lys Thr Ala Val Leu Arg Glu Tyr Glu Ala Leu Lys Gly Leu Arg 2340 2345 2350 cac ccg cac ctg gcc cag ctg cac gca gcc tac ctc agc ccc cgg cac 7163 His Pro His Leu Ala Gln Leu His Ala Ala Tyr Leu Ser Pro Arg His 2355 2360 2365 ctg gtg ctc atc ttg gag ctg tgc tct ggg ccc gag ctg ctc ccc tgc 7211 Leu Val Leu Ile Leu Glu Leu Cys Ser Gly Pro Glu Leu Leu Pro Cys 2370 2375 2380 ctg gcc gag agg gcc tcc tac tca gaa tcc gag gtg aag gac tac ctg 7259 Leu Ala Glu Arg Ala Ser Tyr Ser Glu Ser Glu Val Lys Asp Tyr Leu 2385 2390 2395 2400 tgg cag atg ttg agt gcc acc cag tac ctg cac aac cag cac atc ctg 7307 Trp Gln Met Leu Ser Ala Thr Gln Tyr Leu His Asn Gln His Ile Leu 2405 2410 2415 cac ctg gac ctg agg tcc gag aac atg atc atc acc gaa tac aac ctg 7355 His Leu Asp Leu Arg Ser Glu Asn Met Ile Ile Thr Glu Tyr Asn Leu 2420 2425 2430 ctc aag gtc gtg gac ctg ggc aat gca cag agc ctc agc cag gag aag 7403 Leu Lys Val Val Asp Leu Gly Asn Ala Gln Ser Leu Ser Gln Glu Lys 2435 2440 2445 gtg ctg ccc tca gac aag ttc aag gac tac cta gag acc atg gct cca 7451 Val Leu Pro Ser Asp Lys Phe Lys Asp Tyr Leu Glu Thr Met Ala Pro 2450 2455 2460 gag ctc ctg gag ggc cag ggg gct gtt cca cag aca gac atc tgg gcc 7499 Glu Leu Leu Glu Gly Gln Gly Ala Val Pro Gln Thr Asp Ile Trp Ala 2465 2470 2475 2480 atc ggt gtg aca gcc ttc atc atg ctg agc gcc gag tac ccg gtg agc 7547 Ile Gly Val Thr Ala Phe Ile Met Leu Ser Ala Glu Tyr Pro Val Ser 2485 2490 2495 agc gag ggt gca cgc gac ctg cag aga gga ctg cgc aag ggg ctg gtc 7595 Ser Glu Gly Ala Arg Asp Leu Gln Arg Gly Leu Arg Lys Gly Leu Val 2500 2505 2510 cgg ctg agc cgc tgc tac gcg ggg ctg tcc ggg ggc gcc gtg gcc ttc 7643 Arg Leu Ser Arg Cys Tyr Ala Gly Leu Ser Gly Gly Ala Val Ala Phe 2515 2520 2525 ctg cgc agc act ctg tgc gcc cag ccc tgg ggc cgg ccc tgc gcg tcc 7691 Leu Arg Ser Thr Leu Cys Ala Gln Pro Trp Gly Arg Pro Cys Ala Ser 2530 2535 2540 agc tgc ctg cag tgc ccg tgg cta aca gag gag ggc ccg gcc tgt tcg 7739 Ser Cys Leu Gln Cys Pro Trp Leu Thr Glu Glu Gly Pro Ala Cys Ser 2545 2550 2555 2560 cgg ccc gcg ccc gtg acc ttc cct acc gcg cgg ctg cgc gtc ttc gtg 7787 Arg Pro Ala Pro Val Thr Phe Pro Thr Ala Arg Leu Arg Val Phe Val 2565 2570 2575 cgc aat cgc gag aag aga cgc gcg ctg ctg tac aag agg cac aac ctg 7835 Arg Asn Arg Glu Lys Arg Arg Ala Leu Leu Tyr Lys Arg His Asn Leu 2580 2585 2590 gcc cag gtg cgc tgagggtcgc cccggccaca cccttggtct ccccgctggg 7887 Ala Gln Val Arg 2595 ggtcgctgca gacgcgccaa taaaaacgca cagccgggcg a 7928 6 2596 PRT Homo sapiens 6 Met Leu Glu Arg Phe Thr Pro Lys Lys Val Lys Lys Gly Ser Ser Ile 1 5 10 15 Thr Phe Ser Val Lys Val Glu Gly Arg Pro Val Pro Thr Val His Trp 20 25 30 Leu Arg Glu Glu Ala Glu Arg Gly Val Leu Trp Ile Gly Pro Asp Thr 35 40 45 Pro Gly Tyr Thr Val Ala Ser Ser Ala Gln Gln His Ser Leu Val Leu 50 55 60 Leu Asp Val Gly Arg Gln His Gln Gly Thr Tyr Thr Cys Ile Ala Ser 65 70 75 80 Asn Ala Ala Gly Gln Ala Leu Cys Ser Ala Ser Leu His Val Ser Gly 85 90 95 Leu Pro Lys Val Glu Glu Gln Glu Lys Val Lys Glu Ala Leu Ile Ser 100 105 110 Thr Phe Leu Gln Gly Thr Thr Gln Ala Ile Ser Ala Gln Gly Leu Glu 115 120 125 Thr Ala Ser Phe Ala Asp Leu Gly Gly Gln Arg Lys Glu Glu Pro Leu 130 135 140 Ala Ala Lys Glu Ala Leu Gly His Leu Ser Leu Ala Glu Val Gly Thr 145 150 155 160 Glu Glu Phe Leu Gln Lys Leu Thr Ser Gln Ile Thr Glu Met Val Ser 165 170 175 Ala Lys Ile Thr Gln Ala Lys Leu Gln Val Pro Gly Gly Asp Ser Asp 180 185 190 Glu Asp Ser Lys Thr Pro Ser Ala Ser Pro Arg His Gly Arg Ser Arg 195 200 205 Pro Ser Ser Ser Ile Gln Glu Ser Ser Ser Glu Ser Glu Asp Gly Asp 210 215 220 Ala Arg Gly Glu Ile Phe Asp Ile Tyr Val Val Thr Ala Asp Tyr Leu 225 230 235 240 Pro Leu Gly Ala Glu Gln Asp Ala Ile Thr Leu Arg Glu Gly Gln Tyr 245 250 255 Val Glu Val Leu Asp Ala Ala His Pro Leu Arg Trp Leu Val Arg Thr 260 265 270 Lys Pro Thr Lys Ser Ser Pro Ser Arg Gln Gly Trp Val Ser Pro Ala 275 280 285 Tyr Leu Asp Arg Arg Leu Lys Leu Ser Pro Glu Trp Gly Ala Ala Glu 290 295 300 Ala Pro Glu Phe Pro Gly Glu Ala Val Ser Glu Asp Glu Tyr Lys Ala 305 310 315 320 Arg Leu Ser Ser Val Ile Gln Glu Leu Leu Ser Ser Glu Gln Ala Phe 325 330 335 Val Glu Glu Leu Gln Phe Leu Gln Ser His His Leu Gln His Leu Glu 340 345 350 Arg Cys Pro His Val Pro Ile Ala Val Ala Gly Gln Lys Ala Val Ile 355 360 365 Phe Arg Asn Val Arg Asp Ile Gly Arg Phe His Ser Ser Phe Leu Gln 370 375 380 Glu Leu Gln Gln Cys Asp Thr Asp Asp Asp Val Ala Met Cys Phe Ile 385 390 395 400 Lys Asn Gln Ala Ala Phe Glu Gln Tyr Leu Glu Phe Leu Val Gly Arg 405 410 415 Val Gln Ala Glu Ser Val Val Val Ser Thr Ala Ile Gln Glu Phe Tyr 420 425 430 Lys Lys Tyr Ala Glu Glu Ala Leu Leu Ala Gly Asp Pro Ser Gln Pro 435 440 445 Pro Pro Pro Pro Leu Gln His Tyr Leu Glu Gln Pro Val Glu Arg Val 450 455 460 Gln Arg Tyr Gln Ala Leu Leu Lys Glu Leu Ile Arg Asn Lys Ala Arg 465 470 475 480 Asn Arg Gln Asn Cys Ala Leu Leu Glu Gln Ala Tyr Ala Val Val Ser 485 490 495 Ala Leu Pro Gln Arg Ala Glu Asn Lys Leu His Val Ser Leu Met Glu 500 505 510 Asn Tyr Pro Gly Thr Leu Glu Ala Leu Gly Glu Pro Ile Arg Gln Gly 515 520 525 His Phe Ile Val Trp Glu Gly Ala Pro Gly Ala Arg Met Pro Trp Lys 530 535 540 Gly His Asn Arg His Val Phe Leu Phe Arg Asn His Leu Val Ile Cys 545 550 555 560 Lys Pro Arg Arg Asp Ser Arg Thr Asp Thr Val Ser Tyr Val Phe Arg 565 570 575 Asn Met Met Lys Leu Ser Ser Ile Asp Leu Asn Asp Gln Val Glu Gly 580 585 590 Asp Asp Arg Ala Phe Glu Val Trp Gln Glu Arg Glu Asp Ser Val Arg 595 600 605 Lys Tyr Leu Leu Gln Ala Arg Thr Ala Ile Ile Lys Ser Ser Trp Val 610 615 620 Lys Glu Ile Cys Gly Ile Gln Gln Arg Leu Ala Leu Pro Val Trp Arg 625 630 635 640 Pro Pro Asp Phe Glu Glu Glu Leu Ala Asp Cys Thr Ala Glu Leu Gly 645 650 655 Glu Thr Val Lys Leu Ala Cys Arg Val Thr Gly Thr Pro Lys Pro Val 660 665 670 Ile Ser Trp Tyr Lys Asp Gly Lys Ala Val Gln Val Asp Pro His His 675 680 685 Ile Leu Ile Glu Asp Pro Asp Gly Ser Cys Ala Leu Ile Leu Asp Ser 690 695 700 Leu Thr Gly Val Asp Ser Gly Gln Tyr Met Cys Phe Ala Ala Ser Ala 705 710 715 720 Ala Gly Asn Cys Ser Thr Leu Gly Lys Ile Leu Val Gln Val Pro Pro 725 730 735 Arg Phe Val Asn Lys Val Arg Ala Ser Pro Phe Val Glu Gly Glu Asp 740 745 750 Ala Gln Phe Thr Cys Thr Ile Glu Gly Ala Pro Tyr Pro Gln Ile Arg 755 760 765 Trp Tyr Lys Asp Gly Ala Leu Leu Thr Thr Gly Asn Lys Phe Gln Thr 770 775 780 Leu Ser Glu Pro Arg Ser Gly Leu Leu Val Leu Val Ile Arg Ala Ala 785 790 795 800 Ser Lys Glu Asp Leu Gly Leu Tyr Glu Cys Glu Leu Val Asn Arg Leu 805 810 815 Gly Ser Ala Arg Ala Ser Ala Glu Leu Arg Ile Gln Ser Pro Met Leu 820 825 830 Gln Ala Gln Glu Gln Cys His Arg Glu Gln Leu Val Ala Ala Val Glu 835 840 845 Asp Thr Thr Leu Glu Arg Ala Asp Gln Glu Val Thr Ser Val Leu Lys 850 855 860 Arg Leu Leu Gly Pro Lys Ala Pro Gly Pro Ser Thr Gly Asp Leu Thr 865 870 875 880 Gly Pro Gly Pro Cys Pro Arg Gly Ala Pro Ala Leu Gln Glu Thr Gly 885 890 895 Ser Gln Pro Pro Val Thr Gly Thr Ser Glu Ala Pro Ala Val Pro Pro 900 905 910 Arg Val Pro Gln Pro Leu Leu His Glu Gly Pro Glu Gln Glu Pro Glu 915 920 925 Ala Ile Ala Arg Ala Gln Glu Trp Thr Val Pro Ile Arg Met Glu Gly 930 935 940 Ala Ala Trp Pro Gly Ala Gly Thr Gly Glu Leu Leu Trp Asp Val His 945 950 955 960 Ser His Val Val Arg Glu Thr Thr Gln Arg Thr Tyr Thr Tyr Gln Ala 965 970 975 Ile Asp Thr His Thr Ala Arg Pro Pro Ser Met Gln Val Thr Ile Glu 980 985 990 Asp Val Gln Ala Gln Thr Gly Gly Thr Ala Gln Phe Glu Ala Ile Ile 995 1000 1005 Glu Gly Asp Pro Gln Pro Ser Val Thr Trp Tyr Lys Asp Ser Val Gln 1010 1015 1020 Leu Val Asp Ser Thr Arg Leu Ser Gln Gln Gln Glu Gly Thr Thr Tyr 1025 1030 1035 1040 Ser Leu Val Leu Arg His Val Ala Ser Lys Asp Ala Gly Val Tyr Thr 1045 1050 1055 Cys Leu Ala Gln Asn Thr Gly Gly Gln Val Leu Cys Lys Ala Glu Leu 1060 1065 1070 Leu Val Leu Gly Gly Asp Asn Glu Pro Asp Ser Glu Lys Gln Ser His 1075 1080 1085 Arg Arg Lys Leu His Ser Phe Tyr Glu Val Lys Glu Glu Ile Gly Arg 1090 1095 1100 Gly Val Phe Gly Phe Val Lys Arg Val Gln His Lys Gly Asn Lys Ile 1105 1110 1115 1120 Leu Cys Ala Ala Lys Phe Ile Pro Leu Arg Ser Arg Thr Arg Ala Gln 1125 1130 1135 Ala Tyr Arg Glu Arg Asp Ile Leu Ala Ala Leu Ser His Pro Leu Val 1140 1145 1150 Thr Gly Leu Leu Asp Gln Phe Glu Thr Arg Lys Thr Leu Ile Leu Ile 1155 1160 1165 Leu Glu Leu Cys Ser Ser Glu Glu Leu Leu Asp Arg Leu Tyr Arg Lys 1170 1175 1180 Gly Val Val Thr Glu Ala Glu Val Lys Val Tyr Ile Gln Gln Leu Val 1185 1190 1195 1200 Glu Gly Leu His Tyr Leu His Ser His Gly Val Leu His Leu Asp Ile 1205 1210 1215 Lys Pro Ser Asn Ile Leu Met Val His Pro Ala Arg Glu Asp Ile Lys 1220 1225 1230 Ile Cys Asp Phe Gly Phe Ala Gln Asn Ile Thr Pro Ala Glu Leu Gln 1235 1240 1245 Phe Ser Gln Tyr Gly Ser Pro Glu Phe Val Ser Pro Glu Ile Ile Gln 1250 1255 1260 Gln Asn Pro Val Ser Glu Ala Ser Asp Ile Trp Ala Met Gly Val Ile 1265 1270 1275 1280 Ser Tyr Leu Ser Leu Thr Cys Ser Ser Pro Phe Ala Gly Glu Ser Asp 1285 1290 1295 Arg Ala Thr Leu Leu Asn Val Leu Glu Gly Arg Val Ser Trp Ser Ser 1300 1305 1310 Pro Met Ala Ala His Leu Ser Glu Asp Ala Lys Asp Phe Ile Lys Ala 1315 1320 1325 Thr Leu Gln Arg Ala Pro Gln Ala Arg Pro Ser Ala Ala Gln Cys Leu 1330 1335 1340 Ser His Pro Trp Phe Leu Lys Ser Met Pro Ala Glu Glu Ala His Phe 1345 1350 1355 1360 Ile Asn Thr Lys Gln Leu Lys Phe Leu Leu Ala Arg Ser Arg Trp Gln 1365 1370 1375 Arg Ser Leu Met Ser Tyr Lys Ser Ile Leu Val Met Arg Ser Ile Pro 1380 1385 1390 Glu Leu Leu Arg Gly Pro Pro Asp Ser Pro Ser Leu Gly Val Ala Arg 1395 1400 1405 His Leu Cys Arg Asp Thr Gly Gly Ser Ser Ser Ser Ser Ser Ser Ser 1410 1415 1420 Asp Asn Glu Leu Ala Pro Phe Ala Arg Ala Lys Ser Leu Pro Pro Ser 1425 1430 1435 1440 Pro Val Thr His Ser Pro Leu Leu His Pro Arg Gly Phe Leu Arg Pro 1445 1450 1455 Ser Ala Ser Leu Pro Glu Glu Ala Glu Ala Ser Glu Arg Ser Thr Glu 1460 1465 1470 Ala Pro Ala Pro Pro Ala Ser Pro Glu Gly Ala Gly Pro Pro Ala Ala 1475 1480 1485 Gln Gly Cys Val Pro Arg His Ser Val Ile Arg Ser Leu Phe Tyr His 1490 1495 1500 Gln Ala Gly Glu Ser Pro Glu His Gly Ala Leu Ala Pro Gly Ser Arg 1505 1510 1515 1520 Arg His Pro Ala Arg Arg Arg His Leu Leu Lys Gly Gly Tyr Ile Ala 1525 1530 1535 Gly Ala Leu Pro Gly Leu Arg Glu Pro Leu Met Glu His Arg Val Leu 1540 1545 1550 Glu Glu Glu Ala Ala Arg Glu Glu Gln Ala Thr Leu Leu Ala Lys Ala 1555 1560 1565 Pro Ser Phe Glu Thr Ala Leu Arg Leu Pro Ala Ser Gly Thr His Leu 1570 1575 1580 Ala Pro Gly His Ser His Ser Leu Glu His Asp Ser Pro Ser Thr Pro 1585 1590 1595 1600 Arg Pro Ser Ser Glu Ala Cys Gly Glu Ala Gln Arg Leu Pro Ser Ala 1605 1610 1615 Pro Ser Gly Gly Ala Pro Ile Arg Asp Met Gly His Pro Gln Gly Ser 1620 1625 1630 Lys Gln Leu Pro Ser Thr Gly Gly His Pro Gly Thr Ala Gln Pro Glu 1635 1640 1645 Arg Pro Ser Pro Asp Ser Pro Trp Gly Gln Pro Ala Pro Phe Cys His 1650 1655 1660 Pro Lys Gln Gly Ser Ala Pro Gln Glu Gly Cys Ser Pro His Pro Ala 1665 1670 1675 1680 Val Ala Pro Cys Pro Pro Gly Ser Phe Pro Pro Gly Ser Cys Lys Glu 1685 1690 1695 Ala Pro Leu Val Pro Ser Ser Pro Phe Leu Gly Gln Pro Gln Ala Pro 1700 1705 1710 Pro Ala Pro Ala Lys Ala Ser Pro Pro Leu Asp Ser Lys Met Gly Pro 1715 1720 1725 Gly Asp Ile Ser Leu Pro Gly Arg Pro Lys Pro Gly Pro Cys Ser Ser 1730 1735 1740 Pro Gly Ser Ala Ser Gln Ala Ser Ser Ser Gln Val Ser Ser Leu Arg 1745 1750 1755 1760 Val Gly Ser Ser Gln Val Gly Thr Glu Pro Gly Pro Ser Leu Asp Ala 1765 1770 1775 Glu Gly Trp Thr Gln Glu Ala Glu Asp Leu Ser Asp Ser Thr Pro Thr 1780 1785 1790 Leu Gln Arg Pro Gln Glu Gln Ala Thr Met Arg Lys Phe Ser Leu Gly 1795 1800 1805 Gly Arg Gly Gly Tyr Ala Gly Val Ala Gly Tyr Gly Thr Phe Ala Phe 1810 1815 1820 Gly Gly Asp Ala Gly Gly Met Leu Gly Gln Gly Pro Met Trp Ala Arg 1825 1830 1835 1840 Ile Ala Trp Ala Val Ser Gln Ser Glu Glu Glu Glu Gln Glu Glu Ala 1845 1850 1855 Arg Ala Glu Ser Gln Ser Glu Glu Gln Gln Glu Ala Arg Ala Glu Ser 1860 1865 1870 Pro Leu Pro Gln Val Ser Ala Arg Pro Val Pro Glu Val Gly Arg Ala 1875 1880 1885 Pro Thr Arg Ser Ser Pro Glu Pro Thr Pro Trp Glu Asp Ile Gly Gln 1890 1895 1900 Val Ser Leu Val Gln Ile Arg Asp Leu Ser Gly Asp Ala Glu Ala Ala 1905 1910 1915 1920 Asp Thr Ile Ser Leu Asp Ile Ser Glu Val Asp Pro Ala Tyr Leu Asn 1925 1930 1935 Leu Ser Asp Leu Tyr Asp Ile Lys Tyr Leu Pro Phe Glu Phe Met Ile 1940 1945 1950 Phe Arg Lys Val Pro Lys Ser Ala Gln Pro Glu Pro Pro Ser Pro Met 1955 1960 1965 Ala Glu Glu Glu Leu Ala Glu Phe Pro Glu Pro Thr Trp Pro Trp Pro 1970 1975 1980 Gly Glu Leu Gly Pro His Ala Gly Leu Glu Ile Thr Glu Glu Ser Glu 1985 1990 1995 2000 Asp Val Asp Ala Leu Leu Ala Glu Ala Ala Val Gly Arg Lys Arg Lys 2005 2010 2015 Trp Ser Ser Pro Ser Arg Ser Leu Phe His Phe Pro Gly Arg His Leu 2020 2025 2030 Pro Leu Asp Glu Pro Ala Glu Leu Gly Leu Arg Glu Arg Val Lys Ala 2035 2040 2045 Ser Val Glu His Ile Ser Arg Ile Leu Lys Gly Arg Pro Glu Gly Leu 2050 2055 2060 Glu Lys Glu Gly Pro Pro Arg Lys Lys Pro Gly Leu Ala Ser Phe Arg 2065 2070 2075 2080 Leu Ser Gly Leu Lys Ser Trp Asp Arg Ala Pro Thr Phe Leu Arg Glu 2085 2090 2095 Leu Ser Asp Glu Thr Val Val Leu Gly Gln Ser Val Thr Leu Ala Cys 2100 2105 2110 Gln Val Ser Ala Gln Pro Ala Ala Gln Ala Thr Trp Ser Lys Asp Gly 2115 2120 2125 Ala Pro Leu Glu Ser Ser Ser Arg Val Leu Ile Ser Ala Thr Leu Lys 2130 2135 2140 Asn Phe Gln Leu Leu Thr Ile Leu Val Val Val Ala Glu Asp Leu Gly 2145 2150 2155 2160 Val Tyr Thr Cys Ser Val Ser Asn Ala Leu Gly Thr Val Thr Thr Thr 2165 2170 2175 Gly Val Leu Arg Lys Ala Glu Arg Pro Ser Ser Ser Pro Cys Pro Asp 2180 2185 2190 Ile Gly Glu Val Tyr Ala Asp Gly Val Leu Leu Val Trp Lys Pro Val 2195 2200 2205 Glu Ser Tyr Gly Pro Val Thr Tyr Ile Val Gln Cys Ser Leu Glu Gly 2210 2215 2220 Gly Ser Trp Thr Thr Leu Ala Ser Asp Ile Phe Asp Cys Cys Tyr Leu 2225 2230 2235 2240 Thr Ser Lys Leu Ser Arg Gly Gly Thr Tyr Thr Phe Arg Thr Ala Cys 2245 2250 2255 Val Ser Lys Ala Gly Met Gly Pro Tyr Ser Ser Pro Ser Glu Gln Val 2260 2265 2270 Leu Leu Gly Ala Pro Ser His Leu Ala Ser Glu Glu Glu Ser Gln Gly 2275 2280 2285 Arg Ser Ala Gln Pro Leu Pro Ser Thr Lys Thr Phe Ala Phe Gln Thr 2290 2295 2300 Gln Ile Gln Arg Gly Arg Phe Ser Val Val Arg Gln Cys Trp Glu Lys 2305 2310 2315 2320 Ala Ser Gly Arg Ala Leu Ala Ala Lys Ile Ile Pro Tyr His Pro Lys 2325 2330 2335 Asp Lys Thr Ala Val Leu Arg Glu Tyr Glu Ala Leu Lys Gly Leu Arg 2340 2345 2350 His Pro His Leu Ala Gln Leu His Ala Ala Tyr Leu Ser Pro Arg His 2355 2360 2365 Leu Val Leu Ile Leu Glu Leu Cys Ser Gly Pro Glu Leu Leu Pro Cys 2370 2375 2380 Leu Ala Glu Arg Ala Ser Tyr Ser Glu Ser Glu Val Lys Asp Tyr Leu 2385 2390 2395 2400 Trp Gln Met Leu Ser Ala Thr Gln Tyr Leu His Asn Gln His Ile Leu 2405 2410 2415 His Leu Asp Leu Arg Ser Glu Asn Met Ile Ile Thr Glu Tyr Asn Leu 2420 2425 2430 Leu Lys Val Val Asp Leu Gly Asn Ala Gln Ser Leu Ser Gln Glu Lys 2435 2440 2445 Val Leu Pro Ser Asp Lys Phe Lys Asp Tyr Leu Glu Thr Met Ala Pro 2450 2455 2460 Glu Leu Leu Glu Gly Gln Gly Ala Val Pro Gln Thr Asp Ile Trp Ala 2465 2470 2475 2480 Ile Gly Val Thr Ala Phe Ile Met Leu Ser Ala Glu Tyr Pro Val Ser 2485 2490 2495 Ser Glu Gly Ala Arg Asp Leu Gln Arg Gly Leu Arg Lys Gly Leu Val 2500 2505 2510 Arg Leu Ser Arg Cys Tyr Ala Gly Leu Ser Gly Gly Ala Val Ala Phe 2515 2520 2525 Leu Arg Ser Thr Leu Cys Ala Gln Pro Trp Gly Arg Pro Cys Ala Ser 2530 2535 2540 Ser Cys Leu Gln Cys Pro Trp Leu Thr Glu Glu Gly Pro Ala Cys Ser 2545 2550 2555 2560 Arg Pro Ala Pro Val Thr Phe Pro Thr Ala Arg Leu Arg Val Phe Val 2565 2570 2575 Arg Asn Arg Glu Lys Arg Arg Ala Leu Leu Tyr Lys Arg His Asn Leu 2580 2585 2590 Ala Gln Val Arg 2595 7 871 PRT Homo sapiens 7 Met Gly Pro Gly Asp Ile Ser Leu Pro Gly Arg Pro Lys Pro Gly Pro 1 5 10 15 Cys Ser Ser Pro Gly Ser Ala Ser Gln Ala Ser Ser Ser Gln Val Ser 20 25 30 Ser Leu Arg Val Gly Ser Ser Gln Val Gly Thr Glu Pro Gly Pro Ser 35 40 45 Leu Asp Ala Glu Gly Trp Thr Gln Glu Ala Glu Asp Leu Ser Asp Ser 50 55 60 Thr Pro Thr Leu Gln Arg Pro Gln Glu Gln Ala Thr Met Arg Lys Phe 65 70 75 80 Ser Leu Gly Gly Arg Gly Gly Tyr Ala Gly Val Ala Gly Tyr Gly Thr 85 90 95 Phe Ala Phe Gly Gly Asp Ala Gly Gly Met Leu Gly Gln Gly Pro Met 100 105 110 Trp Ala Arg Ile Ala Trp Ala Val Ser Gln Ser Glu Glu Glu Glu Gln 115 120 125 Glu Glu Ala Arg Ala Glu Ser Gln Ser Glu Glu Gln Gln Glu Ala Arg 130 135 140 Ala Glu Ser Pro Leu Pro Gln Val Ser Ala Arg Pro Val Pro Glu Val 145 150 155 160 Gly Arg Ala Pro Thr Arg Ser Ser Pro Glu Pro Thr Pro Trp Glu Asp 165 170 175 Ile Gly Gln Val Ser Leu Val Gln Ile Arg Asp Leu Ser Gly Asp Ala 180 185 190 Glu Ala Ala Asp Thr Ile Ser Leu Asp Ile Ser Glu Val Asp Pro Ala 195 200 205 Tyr Leu Asn Leu Ser Asp Leu Tyr Asp Ile Lys Tyr Leu Pro Phe Glu 210 215 220 Phe Met Ile Phe Arg Lys Val Pro Lys Ser Ala Gln Pro Glu Pro Pro 225 230 235 240 Ser Pro Met Ala Glu Glu Glu Leu Ala Glu Phe Pro Glu Pro Thr Trp 245 250 255 Pro Trp Pro Gly Glu Leu Gly Pro His Ala Gly Leu Glu Ile Thr Glu 260 265 270 Glu Ser Glu Asp Val Asp Ala Leu Leu Ala Glu Ala Ala Val Gly Arg 275 280 285 Lys Arg Lys Trp Ser Ser Pro Ser Arg Ser Leu Phe His Phe Pro Gly 290 295 300 Arg His Leu Pro Leu Asp Glu Pro Ala Glu Leu Gly Leu Arg Glu Arg 305 310 315 320 Val Lys Ala Ser Val Glu His Ile Ser Arg Ile Leu Lys Gly Arg Pro 325 330 335 Glu Gly Leu Glu Lys Glu Gly Pro Pro Arg Lys Lys Pro Gly Leu Ala 340 345 350 Ser Phe Arg Leu Ser Gly Leu Lys Ser Trp Asp Arg Ala Pro Thr Phe 355 360 365 Leu Arg Glu Leu Ser Asp Glu Thr Val Val Leu Gly Gln Ser Val Thr 370 375 380 Leu Ala Cys Gln Val Ser Ala Gln Pro Ala Ala Gln Ala Thr Trp Ser 385 390 395 400 Lys Asp Gly Ala Pro Leu Glu Ser Ser Ser Arg Val Leu Ile Ser Ala 405 410 415 Thr Leu Lys Asn Phe Gln Leu Leu Thr Ile Leu Val Val Val Ala Glu 420 425 430 Asp Leu Gly Val Tyr Thr Cys Ser Val Ser Asn Ala Leu Gly Thr Val 435 440 445 Thr Thr Thr Gly Val Leu Arg Lys Ala Glu Arg Pro Ser Ser Ser Pro 450 455 460 Cys Pro Asp Ile Gly Glu Val Tyr Ala Asp Gly Val Leu Leu Val Trp 465 470 475 480 Lys Pro Val Glu Ser Tyr Gly Pro Val Thr Tyr Ile Val Gln Cys Ser 485 490 495 Leu Glu Gly Gly Ser Trp Thr Thr Leu Ala Ser Asp Ile Phe Asp Cys 500 505 510 Cys Tyr Leu Thr Ser Lys Leu Ser Arg Gly Gly Thr Tyr Thr Phe Arg 515 520 525 Thr Ala Cys Val Ser Lys Ala Gly Met Gly Pro Tyr Ser Ser Pro Ser 530 535 540 Glu Gln Val Leu Leu Gly Gly Pro Ser His Leu Ala Ser Glu Glu Glu 545 550 555 560 Ser Gln Gly Arg Ser Ala Gln Pro Leu Pro Ser Thr Lys Thr Phe Ala 565 570 575 Phe Gln Thr Gln Ile Gln Arg Gly Arg Phe Ser Val Val Arg Gln Cys 580 585 590 Trp Glu Lys Ala Ser Gly Arg Ala Leu Ala Ala Lys Ile Ile Pro Tyr 595 600 605 His Pro Lys Asp Lys Thr Ala Val Leu Arg Glu Tyr Glu Ala Leu Lys 610 615 620 Gly Leu Arg His Pro His Leu Ala Gln Leu His Ala Ala Tyr Leu Ser 625 630 635 640 Pro Arg His Leu Val Leu Ile Leu Glu Leu Cys Ser Gly Pro Glu Leu 645 650 655 Leu Pro Cys Leu Ala Glu Arg Ala Ser Tyr Ser Glu Ser Glu Val Lys 660 665 670 Asp Tyr Leu Trp Gln Met Leu Ser Ala Thr Gln Tyr Leu His Asn Gln 675 680 685 His Ile Leu His Leu Asp Leu Arg Ser Glu Asn Met Ile Ile Thr Glu 690 695 700 Tyr Asn Leu Leu Lys Val Val Asp Leu Gly Asn Ala Gln Ser Leu Ser 705 710 715 720 Gln Glu Lys Val Leu Pro Ser Asp Lys Phe Lys Asp Tyr Leu Glu Thr 725 730 735 Met Ala Pro Glu Leu Leu Glu Gly Gln Gly Ala Val Pro Gln Thr Asp 740 745 750 Ile Trp Ala Ile Gly Val Thr Ala Phe Ile Met Leu Ser Ala Glu Tyr 755 760 765 Pro Val Ser Ser Glu Gly Ala Arg Asp Leu Gln Arg Gly Leu Arg Lys 770 775 780 Gly Leu Val Arg Leu Ser Arg Cys Tyr Ala Gly Leu Ser Gly Gly Ala 785 790 795 800 Val Ala Phe Leu Arg Ser Thr Leu Cys Ala Gln Pro Trp Gly Arg Pro 805 810 815 Cys Ala Ser Ser Cys Leu Gln Cys Pro Trp Leu Thr Glu Glu Gly Pro 820 825 830 Ala Cys Ser Arg Pro Ala Pro Val Thr Phe Pro Thr Ala Arg Leu Arg 835 840 845 Val Phe Val Arg Asn Arg Glu Lys Arg Arg Ala Leu Leu Tyr Lys Arg 850 855 860 His Asn Leu Ala Gln Val Arg 865 870 8 548 PRT Rattus norvegicus 8 Met Ala His Ile Ser Arg Ile Leu Lys Gly Lys Pro Glu Gly Pro Glu 1 5 10 15 Lys Glu Gly Pro Pro Arg Lys Lys Ala Gly Leu Ala Ser Phe Arg Leu 20 25 30 Ser Gly Leu Lys Gly Arg Asp Gln Ala Pro Ser Phe Leu Arg Glu Leu 35 40 45 Ser Asp Glu Ala Val Val Leu Gly Gln Ser Val Thr Leu Ala Cys Gln 50 55 60 Val Leu Ala Gln Pro Thr Ala Gln Ala Thr Trp Ser Lys Asp Gly Ala 65 70 75 80 Leu Leu Glu Ser Ser Gly His Leu Leu Ile Ser Ser Thr Leu Lys Asn 85 90 95 Phe Gln Leu Leu Thr Ile Leu Val Val Thr Glu Glu Asp Leu Gly Thr 100 105 110 Tyr Thr Cys Cys Val Ser Asn Pro Leu Gly Thr Ala Val Thr Thr Gly 115 120 125 Val Leu Arg Lys Ala Glu Arg Pro Ser Ser Ser Pro Arg Pro Glu Val 130 135 140 Gly Glu Leu Tyr Thr Asp Ala Val Leu Leu Val Trp Lys Pro Val Glu 145 150 155 160 Ser Tyr Gly Pro Val Thr Tyr Ile Val Gln Cys Cys Ile Glu Gly Gly 165 170 175 Ser Trp Thr Thr Leu Ala Ser Asp Ile Ser Asp Cys Cys Tyr Leu Thr 180 185 190 Gly Lys Leu Pro Arg Gly Gly Met Tyr Thr Phe Arg Thr Ala Cys Val 195 200 205 Ser Lys Ala Gly Met Gly Pro Tyr Ser Ser Pro Ser Glu Gln Val Leu 210 215 220 Leu Gly Gly Pro Asn His Leu Ala Ser Glu Glu Glu Ser Ser Arg Gly 225 230 235 240 Arg Pro Ala Gln Leu Leu Pro Ser Thr Lys Thr Phe Ala Phe Gln Thr 245 250 255 Gln Ile Arg Arg Gly Arg Phe Ser Val Val Arg Gln Cys Arg Glu Lys 260 265 270 Ala Ser Gly Arg Ala Leu Ala Ala Lys Ile Val Pro Tyr Gln Pro Glu 275 280 285 Asp Lys Thr Thr Val Leu Arg Glu Tyr Glu Ala Leu Lys Arg Leu His 290 295 300 His Pro His Leu Ala Gln Leu His Ala Ala Tyr Leu Ser Pro Arg His 305 310 315 320 Leu Val Leu Ile Leu Glu Leu Cys Ser Gly Pro Glu Leu Leu Pro Ser 325 330 335 Leu Ala Glu Arg Asp Ser Tyr Ser Glu Ser Asp Val Lys Asp Tyr Leu 340 345 350 Trp Gln Met Leu Ser Ala Thr Gln Tyr Leu His Ala Gln His Ile Leu 355 360 365 His Leu Asp Leu Arg Ser Glu Asn Met Met Val Thr Glu Tyr Asn Leu 370 375 380 Leu Lys Val Ile Asp Leu Gly Asn Ala Gln Ser Leu Ser Gln Glu Lys 385 390 395 400 Val Pro Pro Pro Glu Asn Phe Lys Asp Tyr Leu Glu Thr Met Ala Pro 405 410 415 Glu Leu Leu Glu Gly Gln Gly Ala Val Pro Gln Thr Asp Ile Trp Ala 420 425 430 Ile Gly Val Thr Ala Phe Ile Met Leu Ser Gly Glu Tyr Pro Val Ser 435 440 445 Ser Glu Gly Thr Arg Asp Leu Gln Lys Gly Leu Arg Lys Gly Leu Ile 450 455 460 Gln Leu Ser Arg Cys Tyr Ala Gly Leu Ser Gly Gly Ala Val Ala Phe 465 470 475 480 Leu Gln Ser Ser Leu Cys Ala Arg Pro Trp Gly Arg Pro Cys Ala Ser 485 490 495 Thr Cys Leu Gln Cys Gly Trp Leu Thr Glu Glu Gly Pro Thr Gly Ser 500 505 510 Arg Pro Thr Pro Val Thr Phe Pro Thr Ala Arg Leu Arg Ala Phe Val 515 520 525 Arg Glu Arg Glu Lys Arg Arg Ala Leu Leu Tyr Lys Lys His Asn Leu 530 535 540 Ala Gln Val Arg 545 9 548 PRT Mus musculus 9 Met Ala His Ile Ser Arg Ile Leu Lys Gly Arg Pro Glu Gly Pro Glu 1 5 10 15 Arg Glu Gly Pro Pro Arg Lys Lys Ala Gly Leu Ala Ser Phe Arg Leu 20 25 30 Ser Gly Leu Lys Gly Arg Asp Gln Ala Pro Ser Phe Leu Arg Glu Leu 35 40 45 Ser Asp Glu Ala Val Val Leu Gly Gln Ser Val Thr Leu Ala Cys Gln 50 55 60 Val Leu Ala Gln Pro Thr Ala Gln Ala Thr Trp Ser Lys Asp Gly Val 65 70 75 80 Leu Leu Glu Ser Ser Gly His Leu Leu Ile Ser Ser Thr Leu Lys Asn 85 90 95 Phe Gln Leu Leu Thr Ile Leu Val Val Lys Glu Glu Asp Leu Gly Thr 100 105 110 Tyr Thr Cys Cys Val Ser Asn Pro Leu Gly Thr Ala Val Thr Thr Gly 115 120 125 Val Leu Arg Lys Ala Glu Arg Pro Ser Ser Ser Pro Arg Pro Glu Val 130 135 140 Gly Glu Leu Tyr Lys Asp Ala Val Leu Leu Val Trp Lys Pro Val Glu 145 150 155 160 Ser Cys Gly Pro Val Thr Tyr Ile Val Gln Cys Cys Ile Glu Gly Gly 165 170 175 Ser Trp Thr Thr Leu Ala Ser Asp Ile Ser Asp Cys Cys Tyr Leu Thr 180 185 190 Gly Lys Leu Ser Arg Gly Gly Met Tyr Ile Phe Arg Thr Ala Cys Val 195 200 205 Ser Lys Ala Gly Met Gly Pro Tyr Ser Ser Pro Ser Glu Gln Val Leu 210 215 220 Leu Gly Gly Pro Asn His Leu Ala Ser Glu Glu Glu Ser Ser Arg Gly 225 230 235 240 Arg Pro Ala Gln Leu Leu Pro Ser Thr Lys Thr Phe Ala Phe Gln Met 245 250 255 Gln Ile Arg Arg Gly Arg Phe Ser Val Val Arg Gln Cys Arg Glu Lys 260 265 270 Ala Ser Gly Arg Ala Leu Ala Ala Lys Ile Val Pro Tyr Gln Pro Glu 275 280 285 Asp Lys Thr Ala Val Leu Arg Glu Tyr Glu Ala Leu Lys Arg Leu His 290 295 300 His Pro His Leu Ala Gln Leu His Ala Ala Tyr Leu Ser Pro Arg His 305 310 315 320 Leu Val Leu Ile Leu Glu Leu Cys Ser Gly Pro Glu Leu Leu Pro Ser 325 330 335 Leu Ala Glu Arg Glu Ser Tyr Ser Glu Ser Asp Val Lys Asp Tyr Leu 340 345 350 Trp Gln Met Leu Ser Ala Thr Gln Tyr Leu His Ala Gln His Ile Leu 355 360 365 His Leu Asp Leu Arg Ser Glu Asn Met Met Val Thr Glu Tyr Asn Leu 370 375 380 Leu Lys Val Ile Asp Leu Gly Asn Ala Gln Ser Leu Asp Gln Glu Lys 385 390 395 400 Val Pro Ala Pro Glu Asn Phe Lys Asp Tyr Leu Glu Thr Met Ala Pro 405 410 415 Glu Leu Leu Glu Gly Gln Gly Ala Val Pro Gln Thr Asp Ile Trp Ala 420 425 430 Ile Gly Val Thr Ala Phe Ile Met Leu Ser Gly Glu Tyr Pro Glu Ser 435 440 445 Ser Glu Gly Thr Arg Asp Leu Gln Lys Gly Leu Arg Lys Gly Leu Ile 450 455 460 Arg Leu Ser Arg Cys Tyr Ala Gly Leu Ser Gly Gly Ala Val Ala Phe 465 470 475 480 Leu Gln Ser Ser Leu Cys Ala Gln Pro Trp Gly Arg Pro Cys Ala Ser 485 490 495 Thr Cys Leu Gln Cys Gly Trp Leu Thr Glu Glu Gly Pro Thr Gly Ser 500 505 510 Arg Pro Thr Pro Val Thr Phe Pro Thr Val Arg Leu Arg Ala Phe Val 515 520 525 Arg Glu Arg Glu Lys Arg Arg Ala Leu Leu Tyr Lys Lys His Asn Leu 530 535 540 Ala Gln Val Arg 545 

We claim:
 1. An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3, and 5, wherein the nucleotide sequence encodes a polypeptide capable of catalyzing the transfer of a phosphate group from a donor molecule to an acceptor molecule.
 2. A vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3, and 5, wherein the nucleotide sequence encodes a polypeptide capable of catalyzing the transfer of a phosphate group from a donor molecule to an acceptor molecule.
 3. A host cell transformed with the vector of claim
 2. 4. The host cell of claim 3, wherein said host cell produces a polypeptide capable of catalyzing the transfer of a phosphate group from a donor molecule to an acceptor molecule.
 5. An isolated nucleic acid capable of hybridizing under high stringency conditions to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3, and
 5. 6. The isolated nucleic acid of claim 5, wherein said isolated nucleic acid is capable of inhibiting the regulation of signal transduction.
 7. The isolated nucleic acid of claim 5, wherein said nucleic acid is capable of inhibiting said transfer of said phosphate group from said donor molecule to said acceptor molecule.
 8. A method of preventing or treating disease in a mammal comprising administering to said mammal syngeneic cells transformed with the vector of claim 2, wherein said transformed syngeneic cells produce a polypeptide capable of regulating signal transduction.
 9. The method of claim 8, wherein said mammal is a human.
 10. The method of claim 8, wherein said disease is cardiac disease.
 11. The method of claim 8, wherein the syngeneic cells produces a polypeptide capable of catalyzing the transfer of a phosphate group from a donor molecule to an acceptor molecule.
 12. A method of preventing or treating disease in a mammal comprising administering to said mammal syngeneic cells transformed with the vector of claim 2, wherein said transformed syngeneic cells produce a polypeptide capable of catalyzing the transfer of a phosphate group from a donor molecule to an acceptor molecule.
 13. The method of claim 12, wherein said mammal is a human.
 14. The method of claim 12, wherein said disease is cardiac disease. 